Compounds inducible degradation of sos1 protein, methods of making and uses

Abstract

The application discloses a compound capable of inducing SOS1 protein degradation, a preparation method and purposes, and the structure of the compound is shown as formula I. The compound of the application can induce SOS1 protein degradation, is used as a SOS1 degrading agent, and is used for preventing and / or treating tumors. The SOS1 protein ligand-connecting group L-E3 ligase ligand (I).

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to compounds that can induce the degradation of SOS1 protein, methods for their preparation, pharmaceutical compositions containing such compounds, and their medical uses as SOS1 degrading agents for the prevention and / or treatment of tumors. Background Technology

[0002] Studies have found RAS gene mutations in approximately 30% of tumors, and these mutations play a crucial role in the early formation and development of tumors. Humans can encode three RAS genes: KRAS, HRAS, and NRAS. KRAS mutations account for the largest proportion of all RAS-mutated tumors, approximately 86%. High rates of KRAS mutations have been detected in patients with high-mortality pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma, at 97.7%, 44.7%, and 30.9%, respectively. KRAS mutations lead to the sustained activation of signaling pathways such as RAF-MAPK and PI3K-AKT, thereby affecting tumor cell proliferation, differentiation, and migration. KRAS exists in two states in cells: an activated state bound to GTP and an inactivated state bound to GDP. Two types of proteins in cells are involved in regulating the switching between these two states: guanine nucleotide exchange factors (GEFs) and GTPase activators (GAPs). GEFs can promote the dissociation of KRAS from GDP, thereby accelerating the binding of KRAS to GTP and activating the KRAS protein. The human body contains various KRAS-related GEFs, with SOS (son of sevenless) being an important guanine nucleotide exchange factor. SOS proteins are a class of functional proteins encoded by the SOS gene. Researchers such as Bonfini et al. first discovered these proteins during their study of Drosophila eye development. In humans, there are two structurally similar SOS proteins: SOS1 and SOS2. Currently, the structure and function of SOS1 have been extensively studied and it is considered to play a more significant role. Mutations and overexpression of the SOS1 gene are closely linked to the development of tumors and other diseases. For example, studies have shown that SOS1 plays an important role in the migration and invasion of breast cancer stem cells (Lin, CH et al., Oncotargets Ther 2018, 11, 5419-5428); in HCT15 colon cancer cells, reducing the expression of SOS1 protein can decrease KRAS activation and tumor formation during subcutaneous xenograft transplantation (Depeille, P. et al., Nat Cell Biol 2015, 17(6), 804-815); in addition, silencing the SOS gene in pancreatic cancer cells can also reduce tumor cell proliferation (Jeng, HH et al., Nat Commun 3:1168 doi:10.1038 / ncomms2173(2012)). Animal studies have also shown that knocking out KRAS-carrying cells... G12D The mutation of the SOS1 gene in mice significantly improves the symptoms of acute proliferative myeloma and prolongs the survival of the mice (You, XN et al., Blood 2018, 132(24), 2575-2579).

[0003] Besides cancer, mutations in the SOS gene leading to amino acid sequence mutations in the SOS protein are a significant factor contributing to many other human diseases. Typical examples include Noonan syndrome and Cardiofacio-cutaneous syndrome. Noonan syndrome patients exhibit symptoms such as skeletal and physical abnormalities, as well as heart defects and intellectual disabilities. Cardiofacio-cutaneous syndrome patients present with similar symptoms. Gene mutation screening in clinical patients has revealed point mutations in multiple genes, including KRAS, BRAF, MEK1, MEK2, and SOS1, along with corresponding amino acid mutations in their encoded proteins, leading to overactivation of the RAS / RAF / MEK / ERK pathways. Similarly, type 1 hereditary gingival fibroma is also a disease caused by overactivation of the RAS / RAF / MEK / ERK pathway. This disease is caused by a mutation in the PR domain of the SOS protein, which causes the SOS protein to bind to the cell membrane, thereby spontaneously activating the RAS / RAF / MEK / ERK pathway and promoting cell proliferation (Hart, TC et al., Am J Hum Genet 2002, 70(4), 943-54).

[0004] SOS1 is also a nucleotide exchange factor for RAC and is involved in the activation of RAC (Lu, TY et al., P Natl AcadSci USA 2014, 111(34), 12544-12549).

[0005] In summary, developing regulators of the SOS1 protein holds promise for a novel class of therapeutic drugs. In recent years, several small molecules directly acting on SOS1 have been reported. Yi Zhang et al. obtained a class of SOS1 inhibitors through virtual screening, one of which, NSC-658497, exhibited a high binding activity to SOS1, KB. d =7.0 μM, at high concentrations, it can inhibit the phosphorylation of ERK kinase in cells and inhibit cell proliferation (Evelyn, CR et al., Chem Biol 2014, 21(12), 1618-1628). In another study by the same research group, two SOS1 inhibitors, UC-773587 and UC-857993, were discovered. The binding activity of these two compounds to SOS1 was also at the micromolar level, and they could inhibit the proliferation of pancreatic cancer cells MIA-PaCa-2, but the activity was still relatively weak (Evelyn, CR et al., J Biol Chem 2015, 290(20), 12879-12898).

[0006]

[0007] In 2019, Bayer reported a class of compounds with 4-substituted aminobenzopyrimidine structures. These molecules can effectively inhibit the interaction between SOS1 and KRAS. A representative compound, BAY-293, exhibits high binding activity to SOS1. d =36 nM, but further studies have shown that this compound cannot completely inhibit KRAS-carrying compounds. G12C Phosphorylation of the downstream signaling pathway ERK in mutant Calu-1 cells only achieved a 50% inhibitory level, but its inhibition level was significantly higher than that of KRAS. G12C When irreversible inhibitors are used in combination, they can synergistically inhibit cell proliferation (Hillig, RC et al., P Natl Acad Sci USA 2019, 116(7), 2551-2560). BI also recently reported a class of SOS1 inhibitors, one of which, BI-3406, has a binding constant K with SOS1. d =9.7 nM, and can effectively inhibit the proliferation of KRAS mutant tumor cells (Hofmann, MH et al., Cancer Discov. 2021, 11(1), 142-157). In addition to the SOS1 inhibitors reported above, Stephen W. Fesik's research group also reported some SOS1 agonists (such as compounds 34 and 64) (Abbott, JR et al., Acs Med Chem Lett 2018, 9(9), 941-946.; Abbott, JR et al., J Med Chem 2018, 61(14), 6002-6017.; Abbott, JR et al., J Med Chem 2018, 61(14), 6002-6017). These compounds can promote the binding of RAS protein to GTP. At low concentrations in cells, they can enhance the phosphorylation level of ERK kinase, but at high concentrations, they can inhibit the phosphorylation of ERK kinase. It is evident that there is still a lack of effective clinical drugs in this field, and it is still in the early research stage. There is an urgent need to develop new compounds to confirm the effectiveness of this strategy.

[0008] Induced protein degradation technology has received widespread attention from industry and academia in recent years, as it is believed to bring about a new class of small molecule drugs that can break the traditional mode of action of small molecule drugs. This type of small molecule degrader can be regarded as a bifunctional molecule, with one end binding to the target protein and the other end acting on E3 ligase. This functional molecule can recruit the target protein to the vicinity of the E3 ligase complex, causing ubiquitination of the target protein, and finally using the intracellular proteasome to selectively induce the degradation of the target protein (Chamberlain, PP et al., Nat Chem Biol 2019, 15(10), 937-944). At present, there are no reports of small molecules that target and induce the degradation of SOS1. Summary of the Invention

[0009] The purpose of this invention is to provide a small molecule that causes SOS1 degradation.

[0010] A first aspect of the present invention provides a protein degradation-targeting chimera of general structural formula I, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof:

[0011] SOS1 protein ligand – linker group L-E3 ligase ligand (I).

[0012] In another preferred embodiment, the SOS1 ligand is selected from the group consisting of:

[0013]

[0014] In the formula, "*" represents the R configuration or the S configuration.

[0015] In another preferred embodiment, the E3 ligase ligand is selected from: cereblonE3 ligase ligand, VHLE3 ligase ligand, MDM2 ligase ligand, TRIM24 ligase ligand, TRIM21 ligase ligand, KEAP1 ligase ligand, and IAP ligase ligand.

[0016] In another preferred embodiment, the -E3 ligase ligand is selected from the group consisting of:

[0017]

[0018] In the formula, X is a hydrogen atom or a halogen.

[0019] In another preferred embodiment, L is represented as: -Z 1 -[W 1 -L'-W 2 ] s -Z 2 -

[0020] Among them, Z 1It connects to the SOS1 ligand and is selected from -C(=O)-, -O-, or is not present;

[0021] Z 2 Ligated with E3 ligase ligand, selected from -(CH2) m C(=O)-、-(CH2) m NH-, -O-, or none;

[0022] s is an integer between 1 and 6;

[0023] L' is selected from: -NHCO- or not present;

[0024] W 1 and W 2 Each is independently selected from -(CH2) m -,-Y-(CH2) m -,-(CH2) n -Y-, -O-(CH2) m -O-(CH2) n -,-(CH2) m -O-(CH2CH2O) n -;

[0025] Each m is independently selected from an integer between 0 and 11;

[0026] Each n is independently selected from an integer between 0 and 6;

[0027] Each Y is independently selected from -O- and -S-.

[0028] In another preferred embodiment, the linking group L is selected from:

[0029]

[0030]

[0031]

[0032] In the formula, "*" represents the R configuration or the S configuration.

[0033] In another preferred embodiment, the protein degradation targeting chimera is selected from the group consisting of:

[0034]

[0035]

[0036] In the formula, "*" indicates the R configuration or S configuration, and X is a hydrogen atom or a halogen. In another preferred embodiment, the SOS1 ligand is selected from the corresponding structures in the above general formula compounds. In another preferred embodiment, the E3 ligase ligand is selected from the corresponding structures in the above general formula compounds.

[0037] In another preferred embodiment, the protein degradation targeting chimera is selected from the group consisting of:

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] In another preferred embodiment, the SOS1 ligand is selected from the corresponding structures of the specific compounds described above. In yet another preferred embodiment, the E3 ligase ligand is selected from the corresponding structures of the specific compounds described above.

[0047] A second aspect of the present invention provides a pharmaceutical composition comprising a compound of formula I as described in the first aspect or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, and a pharmaceutically acceptable carrier.

[0048] In another preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable excipient. In another preferred embodiment, the excipient is selected from fillers, binders, and lubricants. Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula I.

[0049] In some embodiments, the pharmaceutical composition may be used alone or in combination with other types of drugs.

[0050] The other types of drugs mentioned are selected from:

[0051] (1) Inhibitors of EGFR and / or its mutants

[0052] a. For example, afatinib, gefitinib, erlotinib, lapatinib, panitumumab, cetuximab, nimotinib, osimertinib, EGF-816;

[0053] b. The preferred options are afatinib, cetuximab, and osimertinib;

[0054] c. The optimal choice is afatinib.

[0055] (2) Inhibitors of Her2 and / or its mutants

[0056] a. For example, afatinib, trastuzumab, lapatinib, pertuzumab;

[0057] b. Afatinib and trastuzumab are preferred.

[0058] c. Trastuzumab is the optimal choice;

[0059] (3) Inhibitors of ALK and / or its mutants

[0060] a. For example, alectinib, entrectinib, crizotinib, brigatinib;

[0061] b. Alectinib and crizotinib are preferred.

[0062] c. Crizotinib is the preferred choice;

[0063] (4) Inhibitors of MEK and / or its mutants

[0064] a. For example, cobimetinib, bimetinib, trametinib, sermetinib, remdesivir;

[0065] b. Cobimetinib and Trametinib are preferred.

[0066] c. Trametinib is the preferred choice;

[0067] (5) Inhibitors of KRAS G12C

[0068] a. For example, sotorasib, adagrasib, ARS-3248, GDC-6036, AZD-4785;

[0069] (6) BCR-ABL and / or its mutant inhibitors

[0070] a. For example, nilotinib, imatinib, and dasatinib;

[0071] b. Nilotinib and imatinib are preferred;

[0072] c. Imatinib is the optimal choice;

[0073] (7) Inhibitors of c-MET and / or its mutants

[0074] (8). Taxane

[0075] a. For example, paclitaxel, docetaxel;

[0076] b. Paclitaxel is preferred;

[0077] (9) Platinum-containing compounds

[0078] a. For example, cisplatin, oxaliplatin, and carboplatin;

[0079] (10) Antimetabolites

[0080] a. For example, 5-fluorouracil, fluorouridine, gemcitabine, capecitabine;

[0081] b. Preferred gemcitabine;

[0082] (11) Mitotic kinase inhibitors

[0083] a. For example, CDK4 / 6 inhibitors, including abecilibi, palbociclib, and ribociclib;

[0084] b. Abecili and palbocicini are preferred;

[0085] c. The optimal choice is abecili;

[0086] (12). Immunotherapy agents

[0087] a. For example, immune checkpoint inhibitors

[0088] i. For example, anti-CTLA-4 monoclonal antibody, anti-PD1 monoclonal antibody, anti-PD-L1 monoclonal antibody, anti-PD-L2 monoclonal antibody, anti-LAG3 monoclonal antibody, and anti-TIM3 monoclonal antibody;

[0089] ii. Anti-PD1 monoclonal antibodies are preferred;

[0090] iii. For example, iprimma, nivolumab, avelumab, duvalumab, atelizumab, pildizumab, and antipembrolizumab;

[0091] iv. Nivolumab and pembrolizumab are preferred.

[0092] v. The optimal choice is pembrolizumab;

[0093] (13). Anti-angiogenic drugs

[0094] a. For example, bevacizumab, nintedanib;

[0095] b. Bevacizumab is the preferred choice;

[0096] (14) Topoisomerase inhibitors

[0097] a. For example, topotecan and irinotecan;

[0098] b. Irinotecan is the preferred choice;

[0099] (15). Inhibitors of A-Raf and / or B-Raf and / or their mutants

[0100] a. For example, RAF-709, LY-3009120

[0101] (16) ERK and / or its mutant inhibitors

[0102] a. For example, urolithinib;

[0103] (17). Apoptosis regulators

[0104] a. For example, inhibitors of the interaction between p53 and MDM2;

[0105] i. For example, RG-7388, HDM-201, RG-7112, MK-8242, SAR405838, AMG-232, DS-3032;

[0106] ii. Preferred models are RG-7388, HDM-201, and AMG-232.

[0107] b. For example, PARP inhibitors;

[0108] c. For example, MCL-1 inhibitors;

[0109] (18) mTOR inhibitors

[0110] a. For example, rapamycin, everolimus, tesiromolimus, and desfomol;

[0111] (19) Epigenetic regulators

[0112] a. For example, BET inhibitors

[0113] i. For example, JQ-1, OTX 015, GSK 525762, TEN-010;

[0114] b. For example, CDK9 inhibitors;

[0115] (20) Inhibitors of IGF1 / 2 and / or IGF1-R

[0116] a. For example, talutuzumab, MEDI-573.

[0117] A third aspect of the invention provides the use of compounds of general formula I described in the first aspect, and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates, or deuterated compounds thereof, in the preparation of medicaments for diseases associated with SOS1.

[0118] In another preferred embodiment, the diseases associated with SOS1 are selected from: hereditary diseases and cancer.

[0119] The hereditary diseases mentioned are selected from: cardiofacial skin syndrome, hereditary gingival fibromatosis type 1, and Noonan syndrome.

[0120] The cancers mentioned are selected from: colon cancer, lymphoma, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, hepatocellular carcinoma, prostate cancer, breast cancer, bile duct and gallbladder cancer, bladder cancer; brain tumors, including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma; cervical cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcomas.

[0121] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0122] Figure 1 The example shows that compound ZZ02151 in Example 1 can induce the degradation of SOS1 protein in NCI-H358 cells in a dose-dependent manner.

[0123] Figure 2 The example of compound ZC79070 in Example 45 shows that it can induce the degradation of SOS1 protein in NCI-H358 cells in a dose-dependent manner.

[0124] Figure 3 The half-maximal concentration (DC) of compound ZC79070 in Example 45 for the degradation of SOS1 protein in NCI-H358 cells is shown. 50 And the maximum degree of degradation.

[0125] Figure 4 The figure shows the results of the study on the antiproliferative activity of compound ZZ02151 against H358 cells in Example 1.

[0126] Figure 5 The figure shows the results of the study on the antiproliferative activity of compound ZC79070 against H358 cells in Example 45. Detailed Implementation

[0127] Through extensive and in-depth research, the inventors of this application designed and synthesized a class of small molecules that can cause SOS1 degradation by introducing E3 ligase ligands into the solvent-directing region of the SOS1 regulator molecule. Cellular activity tests and degradation mechanism studies were conducted on these molecules, laying the foundation for the development of drugs for the treatment of diseases related to dysfunction of SOS1, RAS, or RAC proteins.

[0128] the term

[0129] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0130] In this invention, the halogen is F, Cl, Br or I.

[0131] The pharmaceutically acceptable salts described in this invention can be salts formed by anion and a positively charged group on a compound of Formula I. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate. Similarly, salts can be formed by cations and negatively charged groups on a compound of Formula I. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium ions.

[0132] In another preferred embodiment, "pharmaceutically acceptable salt" refers to a salt formed by a compound of Formula I with an acid selected from the group consisting of: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid; or a sodium, potassium, calcium, aluminum, or ammonium salt formed by a compound of Formula I with an inorganic base; or a methylamine, ethylamine, or ethanolamine salt formed by a compound of general Formula I with an organic base.

[0133] Pharmaceutical Composition

[0134] The present invention also provides a pharmaceutical composition comprising an active ingredient within a safe and effective range, and a pharmaceutically acceptable carrier.

[0135] The "active ingredient" mentioned in this invention refers to the compound of formula I described in this invention.

[0136] The "active ingredient" and pharmaceutical composition described in this invention can be used as an SOS1 degrading agent to induce the degradation of SOS1 protein for the prevention and / or treatment of cancer, selected from: colon cancer, diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, hepatocellular carcinoma, prostate cancer, breast cancer, bile duct and gallbladder cancer, bladder cancer; brain tumors, including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma; cervical cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcomas. It can also be used to treat hereditary diseases, selected from: cardiofacial skin syndrome, hereditary gingival fibromatosis type 1, and Noonan syndrome.

[0137] "Safe and effective dose" refers to an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg of active ingredient per dose. Preferably, "one dose" refers to one tablet.

[0138] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0139] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0140] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0141] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0142] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0143] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0144] The compounds of this invention can be administered alone or in combination with other therapeutic agents (such as antitumor drugs).

[0145] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0146] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0147] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0148] Example 1

[0149] (2S,4R)-1-((S)-2-(7-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-7-oxohepamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0150]

[0151] Step 1: 7-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutyrate-2-yl)amino)-7-oxoheptanoic acid

[0152]

[0153] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (100 mg, 0.21 mmol), pimelic acid (83.2 mg, 0.52 mmol), EDCI (47.8 mg, 0.25 mmol), and HOAT (5.66 mg, 0.04 mmol) were dissolved in a mixture of N,N-dimethylformamide (4.5 mL) and dichloromethane (0.3 mL). N-methylmorpholine (114 μL, 1.04 mmol) was added under ice bath conditions, and the mixture was stirred for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative HPLC and freeze-dried to obtain 7-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutyrate-2-yl)amino)-7-oxoheptanoic acid (63 mg, yield: 52%).

[0154] Step 2: (2S,4R)-1-((S)-2-(7-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-7-oxohepamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0155]

[0156] (R)-N-(1-(3-(3-amino-5-(trifluoromethyl)phenyl)ethyl)-7-methoxy-2-methyl-6-(piperidin-4-yl)quinazolin-4-amine (50 mg, 0.11 mmol), 7-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutyrate-2-yl)amino)-7-oxoheptanoic acid (64 mg, 0.11 mmol), and HATU (50 mg, 0.13 mmol) were dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (90 μL, 0.55 μL) was then added under ice bath conditions. The mixture was heated and stirred for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to give (2S,4R)-1-((S)-2-(7-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-7-oxohepamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (30 mg, yield 27%).

[0157] 1H NMR(500MHz, Methanol-d4)δ8.97(s,1H),8.30(s,1H),7.45(d,J=8.4Hz,2H),7.42( d,J=8.4Hz,2H),7.11(s,1H),7.07(s,1H),7.04(s,1H),6.97(s,1H),5.79(dt,J=8. 7,6.2Hz,1H),5.03–4.95(m,1H),4.78–4.69(m,1H),4.62(s,1H),4.61–4.53(m,1H) ,4.43(s,1H),4.17–4.08(m,1H),4.04(s,3H),3.87(d,J=11.0Hz,1H),3.74(dd,J=11 .0,3.2Hz,1H),3.39(t,J=12.1Hz,1H),3.30–3.22(m,1H),2.76(t,J=12.7Hz,1H),2 .63(s,3H),2.48(s,3H),2.47–2.43(m,2H),2.42–2.40(m,1H),2.36–2.31(m,1H),2 .30–2.24(m,1H),2.19(t,J=7.6Hz,1H),2.07–1.89(m,4H),1.71(d,J=7.1Hz,3H),1 .68–1.59(m,4H),1.50(d,J=6.9,1.2Hz,3H),1.45–1.37(m,2H),1.04(s,9H).MS-ESI m / z=1028.4[M+1] + .

[0158] Example 2

[0159] (2R,4R)-1-((R)-2-(7-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-7-oxoheptamethamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0160]

[0161] Referring to Example 1, ZZ77061 can be obtained using the same method.

[0162] 1H NMR(500MHz,Methanol-d4)δ8.95(d,J=11.9Hz,1H),8.30(s,1H),7.47–7.40(m,4H ),7.14–6.99(m,3H),6.98–6.88(m,1H),5.78(d,J=7.2Hz,1H),5.38–5.30(m,1H), 5.04–4.99(m,1H),4.74(d,J=12.9Hz,1H),4.50(s,1H),4.46(dd,J=9.3,4.6Hz,1H ),4.36(t,J=4.6Hz,1H),4.14(s,1H),4.07–3.95(m,4H),3.65(d,J=10.4Hz,1H),3 .43–3.35(m,1H),3.25(d,J=13.1Hz,1H),2.75(t,J=12.8Hz,1H),2.63(d,J=1.5Hz ,3H),2.51–2.36(m,5H),2.35–2.24(m,1H),2.19(t,J=7.6Hz,1H),2.03(d,J=6.4H z,1H),1.95–1.85(m,1H),1.70(d,J=7.1Hz,3H),1.64(d,J=7.5Hz,3H),1.49(d,J= 7.0Hz,3H),1.40(d,J=7.6Hz,1H),1.35–1.26(m,5H),1.04(d,J=2.1Hz,9H).MS-ESI m / z=1028.4[M+1] + .

[0163] Example 3

[0164] (2S,4R)-1-((S)-2-(8-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-8-oxoctylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0165]

[0166] Referring to Example 1, ZZ77076 can be obtained using the same method.

[0167] 1H NMR(500MHz,Methanol-d4)δ9.00(d,J=8.9Hz,1H),8.89(d,J=5.7Hz,2H),8.67(ddt,J=7 .9,6.5,1.5Hz,1H),8.30(s,1H),8.12(t,J=6.9Hz,2H),7.45(d,J=8.4Hz,2H),7.42(d,J =8.5Hz,2H),7.18(d,J=23.3Hz,2H),7.06(q,J=6.0,4.2Hz,2H),5.79(q,J=7.1Hz,1H),4 .74(d,J=13.4Hz,1H),4.62(s,1H),4.57(t,J=8.1Hz,1H),4.42(tt,J=4.2,2.0Hz,1H),4. 13(d,J=13.9Hz,1H),4.04(s,3H),3.87(d,J=11.1Hz,1H),3.74(dd,J=11.0,3.9Hz,1H), 3.43–3.35(m,1H),3.25(d,J=13.0Hz,1H),2.80–2.71(m,1H),2.63(s,3H),2.53–2.41(m ,4H),2.30(tq,J=14.0,7.1,6.7Hz,2H),2.20(dd,J=12.9,7.8Hz,1H),2.05–1.87(m,2H) ,1.73–1.70(m,4H),1.68–1.59(m,6H),1.50(d,J=7.0Hz,3H),1.07–1.03(m,10H).MS-ESI m / z=1042.5[M+1] + .

[0168] Example 4

[0169] (2S,4R)-1-((S)-2-(10-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-10-oxododecyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0170]

[0171] Referring to Example 1, ZZ77077 can be obtained using the same method. MS-ESI m / z = 1070.5 [M+1] + .

[0172] Example 5

[0173] (2S,4R)-1-((S)-2-(11-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-11-oxadodecane)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0174]

[0175] Referring to Example 1, ZZ77078 can be obtained using the same method. MS-ESI m / z = 1084.5 [M+1] + .

[0176] Example 6

[0177] (2S,4R)-4-hydroxy-1-((S)-2-(10-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-10-oxododecanoamide)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0178]

[0179] Referring to Example 1, ZZ49071 can be obtained using the same method. 1H NMR(500MHz,DMSO-d6)δ9.54(s,1H),8.99(s,1H),8.52(s,2H),8.31(d,J=7.8Hz,1H),7. 83(d,J=8.9Hz,1H),7.44(d,J=8.3Hz,2H),7.39(d,J=8.3Hz,2H),6.87(d,J=1.7Hz,1H), 6.81(d,J=6.8Hz,2H),6.56(d,J=1.8Hz,1H),5.11(s,2H),4.92(t,J=7.1Hz,1H),4.45(d ,J=8.8Hz,1H),4.41(d,J=13.2Hz,2H),4.31(dd,J=8.6,6.3Hz,1H),4.24(s,3H),4.19(t ,J=5.7Hz,1H),4.13(t,J=6.3Hz,2H),3.60(d,J=11.7Hz,3H),3.38(dd,J=10.2,5.4Hz,1 H),3.24–3.12(m,4H),3.07(t,J=12.5Hz,2H),2.45(s,3H),2.27(ddd,J=29.7,13.0,7.1 Hz,2H),2.16(d,J=2.0Hz,6H),2.10(dt,J=12.2,6.2Hz,1H),1.72–1.63(m,4H),1.48(dt ,J=15.2,7.4Hz,1H),1.38(d,J=7.0Hz,3H),1.26(d,J=31.2Hz,7H),0.95(s,9H).MS-ESI m / z=1055.5[M+1] + .

[0180] Example 7

[0181] (2S,4R)-4-hydroxy-1-((S)-2-(12-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-12-oxododecanoamide)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0182]

[0183] Referring to Example 1, ZZ02030 can be obtained using the same method.

[0184] 1H NMR (500MHz, DMSO-d6) δ9.88(d,J=7.5Hz,1H),8.98(s,1H),8.36(d,J=7.8Hz,1H),8.32(s,1H),7.82(d ,J=2.1Hz,1H),7.78(s,1H),7.76(d,J=2.8Hz,1H),7.66(d,J=7.7Hz,1H),7.62(t,J=7.7Hz,1H),7.44– 7.41(m,2H),7.39–7.36(m,2H),7.07(s,1H),5.85(p,J=7.3Hz,1H),5.32(t,J=5.0Hz,1H),5.09(s,1H) ,4.91(p,J=7.2Hz,1H),4.64(d,J=13.2Hz,1H),4.51(d,J=9.3Hz,1H),4.41(t,J=8.0Hz,1H),4.27(q,J =3.5,3.0Hz,1H),4.02(d,J=14.6Hz,1H),3.99(s,3H),3.66–3.54(m,2H),3.24(t,J=11.8Hz,2H),3.14 (t,J=12.9Hz,2H),2.67–2.60(m,1H),2.56(s,3H),2.45(s,3H),2.36(p,J=1.8Hz,1H),2.33–2.29(m,1 H),2.29–2.24(m,1H),2.24–2.20(m,1H),2.08(dt,J=13.7,6.9Hz,1H),1.82–1.75(m,2H),1.69(d,J=7 .1Hz,3H),1.48(dt,J=25.2,8.9Hz,8H),1.37(d,J=7.0Hz,3H),1.29–1.18(m,8H),0.92(s,9H).MS-ESI m / z=1083.5[M+1] + .

[0185] Example 8

[0186] (2S,4R)-4-hydroxy-1-((S)-2-(7-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-7-oxoheptamethamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0187]

[0188] Referring to Example 1, ZZ02080 can be obtained using the same method. MS-ESI m / z = 1013.5 [M+1] + .

[0189] Example 9

[0190] (2S,4R)-4-hydroxy-1-((S)-2-(8-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-8-oxoctylamino)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0191]

[0192] Referring to Example 1, ZZ02083 can be obtained using the same method. MS-ESI m / z = 1027.5 [M+1] + .

[0193] Example 10

[0194] (2S,4R)-4-hydroxy-1-((S)-2-(9-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-9-oxonamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0195]

[0196] Referring to Example 1, ZZ49107 can be obtained using the same method. MS-ESI m / z = 1041.5 [M+1] + .

[0197] Example 11

[0198] (2S,4R)-4-hydroxy-1-((S)-2-(11-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)-11-oxadodecane)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0199]

[0200] Referring to Example 1, ZZ02038 can be obtained using the same method. MS-ESI m / z = 1069.5 [M+1] + .

[0201] Example 12

[0202] (2S,4R)-4-hydroxy-1-((S)-2-(2-(3-(2-(4-(7-methoxy-2-methyl-4-(((R]-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-2-oxoethoxy)propoxy)acetamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0203]

[0204] Referring to Example 1, ZZ02103 can be obtained using the same method. MS-ESIm / z = 1045.5 [M+1] + .

[0205] Example 13

[0206] (2S,4R)-1-((S)-2-(9-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-9-oxonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0207] Referring to Example 1, ZZ02102 can be obtained using the same method.

[0208]

[0209] 1H NMR(500MHz, Methanol-d4)δ8.97(s,1H),8.29(s,1H),7.45(d,J=8.4Hz,2H),7.42( d,J=8.4Hz,2H),7.09–7.03(m,2H),7.03(s,1H),6.94–6.91(m,1H),5.79(dd,J=7.0 ,3.5Hz,1H),5.00(q,J=7.1Hz,1H),4.74(d,J=13.3Hz,1H),4.62(s,1H),4.57(td,J =7.8,7.0,3.9Hz,1H),4.43(s,1H),4.13(d,J=14.1Hz,1H),4.04(d,J=2.4Hz,3H),3 .88(d,J=11.1Hz,1H),3.74(dd,J=11.0,3.9Hz,1H),3.69–3.66(m,1H),3.57–3.55( m,1H),3.43–3.35(m,1H),3.29–3.22(m,1H),2.75(t,J=12.4Hz,1H),2.63(d,J=1.2 Hz,3H),2.48(d,J=1.5Hz,3H),2.34–2.24(m,2H),1.98–1.88(m,2H),1.71(d,J=7.0 Hz,3H),1.67–1.60(m,8H),1.51(d,J=7.0Hz,3H),1.37(s,6H),1.04(s,9H).MS-ESI m / z=1056.5[M+1] + .

[0210] Example 14

[0211] (2S,4R)-1-((S)-2-(5-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7)-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-5-oxopentanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0212] Referring to Example 1, ZZ02150 can be obtained using the same method.

[0213]

[0214] 1H NMR (500MHz, Methanol-d4) δ9.00 (s, 1H), 8.31 (d, J = 2.4Hz, 1H), 7.47–7.39 (m, 4H),7.20–7.09(m,2H),7.05(s,1H),7.03–6.93(m,1H),5.79(q,J=6.9Hz,1H),5 .03–4.98(m,1H),4.74(d,J=13.6Hz,1H),4.62(d,J=2.6Hz,1H),4.56(dt,J=8.3 ,4.3Hz,1H),4.44–4.41(m,1H),4.13(d,J=13.6Hz,1H),4.04(s,3H),3.99(d,J= 9.8Hz,1H),3.87(s,1H),3.75(s,1H),3.39(td,J=12.1,3.3Hz,1H),3.26(t,J= 13.1Hz,1H),2.83–2.71(m,1H),2.63(s,3H),2.62(d,J=3.1Hz,1H),2.50–2.46( m,5H),2.19(t,J=7.5Hz,2H),1.95–1.89(m,2H),1.72(d,J=7.0Hz,3H),1.70–1. 64(m,2H),1.63–1.56(m,2H),1.49(dd,J=14.3,7.0Hz,3H),1.05(s,9H).MS-ESI m / z = 1000.5[M+1] + .

[0215] Example 15

[0216] (2S,4R)-1-((S)-2-(6-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7)-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-6-oxohexamidinyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl)-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0217] Referring to Example 1, ZZ02152 can be obtained using the same method.

[0218]

[0219] 1H NMR(500MHz, Methanol-d4)δ9.02(d,J=3.8Hz,1H),8.31(d,J=2.0Hz,1H),7.45( d,J=8.4Hz,2H),7.42(d,J=8.4Hz,2H),7.17(s,2H),7.05(d,J=1.4Hz,1H),7.02( s,1H),5.80(q,J=7.1Hz,1H),4.99(d,J=7.1Hz,1H),4.74(d,J=13.3Hz,1H),4.6 2(d,J=3.1Hz,1H),4.57(ddd,J=9.2,7.8,2.0Hz,1H),4.42(dt,J=4.5,2.3Hz,1H) ,4.14(d,J=13.6Hz,1H),4.04(s,4H),3.85(t,J=12.0Hz,1H),3.74(dt,J=10.9, 3.7Hz,1H),3.44–3.35(m,1H),3.27(t,J=14.0Hz,1H),2.76(t,J=12.7Hz,1H),2. 63(s,3H),2.49(s,3H),2.19(t,J=7.6Hz,2H),1.93(td,J=8.8,4.3Hz,2H),1.74– 1.69(m,3H),1.69–1.64(m,8H),1.50(dd,J=7.0,2.3Hz,4H),1.04(s,9H).MS-ESI m / z=1014.5[M+1] + .

[0220] Example 16

[0221] (2S,4R)-4-hydroxy-1-((S)-2-(9-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)nonamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0222]

[0223] Step 1: (2S,4R)-1-((S)-2-(9-bromoaminobenzo)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0224]

[0225] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (100 mg, 0.21 mmol), 9-bromononanoic acid (54 mg, 0.23 mmol), and HATU (87 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (137 μL, 0.83 mmol) was added under ice bath conditions, and the mixture was stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to obtain (2S,4R)-1-((S)-2-(9-bromoaminobenzo)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (86 mg, yield 62%).

[0226] Step 2: (2S,4R)-4-hydroxy-1-((S)-2-(9-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)nonamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0227]

[0228] (2S,4R)-1-((S)-2-(9-bromoaminobenzo)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (75 mg, 0.11 mmol) and (R)-7-methoxy-2-methyl-6-(piperidin-4-yl)-N-(1-(3-(trifluoromethyl)phenyl)ethyl)quinazolin-4-amine (50 mg, 0.11 mmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (93 μL, 0.56 mmol) was added. The mixture was stirred at 60 °C for 16 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to obtain (2S,4R)-4-hydroxy-1-((S)-2-(9-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-ylpiperidin-1-yl)nonamido)-3,3-dimethylbutyryl)-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (43 mg, yield 37%).

[0229] 1H NMR(500MHz,DMSO-d6)δ9.28(s,1H),8.99(s,1H),8.37(d,J=7.8Hz,1H),8.27(s,1H ),7.82(s,1H),7.81–7.76(m,2H),7.67(d,J=7.7Hz,1H),7.62(t,J=7.7Hz,1H),7.4 3(d,J=8.1Hz,2H),7.38(d,J=8.1Hz,2H),7.11(s,1H),5.88(p,J=7.0Hz,1H),5.11( s,1H),4.91(p,J=7.0Hz,1H),4.52(d,J=9.4Hz,1H),4.41(t,J=8.1Hz,1H),4.28(s,1 H),3.99(s,3H),3.61(td,J=10.4,9.9,3.1Hz,4H),3.52–3.46(m,3H),3.23–3.16(m ,2H),3.15–3.05(m,4H),2.58(s,3H),2.45(s,3H),2.27(dt,J=14.8,7.6Hz,1H),2.1 5–2.05(m,4H),2.03–1.91(m,4H),1.79(ddd,J=12.9,8.5,4.6Hz,1H),1.70(d,J=7. 0Hz,3H),1.68(s,2H),1.57–1.43(m,4H),1.37(d,J=7.0Hz,3H),0.94(s,9H).MS-ESI m / z=1027.5[M+1] + .

[0230] Example 17

[0231] 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(3-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethyl)aminodihydroindole-1,3-dione

[0232] Step 1: 3-(2-(2-(((2-(2,6-dioxadiazine-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)tert-butyl propionate

[0233]

[0234] 3-(2-(2-(2-aminoethoxy)ethoxy)propionate tert-butyl ester (100 mg, 0.43 mmol) and 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindole-1,3-dione (130 mg, 0.47 mmol) were dissolved in dimethyl sulfoxide (10 mL), and N,N-diisopropylethylamine (354 μL, 2.14 mmol) was added. The mixture was stirred at 80 °C for 12 hours. Water was added to the reaction mixture. Extracted with ethyl acetate, the organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-TLC (dichloromethane / methanol = 10 / 1) to give tert-butyl 3-(2-(2-(((2-(2,6-dioxadiepidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)propionate (156 mg, yield 74%).

[0235] Step 2: 3-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl-amino)ethoxy)ethoxy)propionic acid

[0236]

[0237] 100 mg (0.20 mmol) of tert-butyl 3-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propionate was dissolved in dichloromethane (4 mL). A solution of dioxane (4 mL, 16 mmol) was added dropwise under ice bath conditions. The mixture was stirred at room temperature for 12 hours. After concentration under reduced pressure, crude 3-(2-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindolin-4-yl-amino)ethoxy)ethoxy)propionate was used directly in the next reaction step.

[0238] Step 3: 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(3-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethyl)aminodihydroindole-1,3-dione

[0239]

[0240] (R)-7-methoxy-2-methyl-6-(piperidin-4-yl)-N-(1-(3-(trifluoromethyl)phenyl)ethyl)quinazolin-4-amine (25 mg, 0.06 mmol), 3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl-amino)ethoxy)ethoxy)propionic acid (24 mg, 0.06 mmol), and HATU (26 mg, 0.07 mmol) were dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (47 μL, 0.28 mmol) was added under ice bath conditions, and the mixture was kept warm and stirred. 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to give 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(3-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethyl)aminodihydroindole-1,3-dione (23 mg, yield 47%).

[0241] 1 H NMR (500MHz, DMSO-d6) δ11.10(s,1H),9.88(d,J=7.6Hz,1H),8.30(s,1H),7.82(s,1H),7.77(d,J=7.7Hz,1H),7.66(d,J=7.7Hz ,1H),7.62(t,J=7.7Hz,1H),7.54(q,J=8.0Hz,1H),7.09–7.00(m,3H),6.59–6.53(m,1H),5.84(q,J=6.7Hz,1H),5.04(dd,J=12. 9,5.5Hz,1H),4.63(d,J=12.7Hz,1H),4.03(d,J=13.3Hz,1H),3.98(s,3H),3.64(t,J=6.6Hz,2H),3.60–3.51(m,8H),3.40(s,3H ),3.24(s,2H),3.12(t,J=13.2Hz,1H),2.87(t,J=17.1Hz,1H),2.65–2.54(m,7H),1.85–1.74(m,2H),1.72–1.62(m,3H).MS-ESI m / z=860.5[M+1] + .

[0242] Example 18

[0243] 2-(2,6-dioxopiperidin-3-yl)-4-((15-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenylethyl)amino)quinazolin-6-yl)-3,6-dihydropyridin-1-1(2H)-yl)-15-oxo-3,6,9,12-tetraoxopentapentadecanyl)aminodihydroindole-1,3-dione

[0244] Referring to Example 17, RX70060 can be obtained using the same method.

[0245]

[0246] 1 H NMR(500MHz,Chloroform-d)δ9.08–8.75(m,2H),8.04(s,1H),7.72(s,1H),7.66(s,1H),7.50(d,J=7.3 Hz,1H),7.48–7.32(m,4H),7.01(dt,J=7.7,3.9Hz,1H),6.87(d,J=8.3Hz,1H),5.79(d,J=56.5Hz,2H),4 .89(dd,J=12.0,6.0Hz,1H),4.34–4.02(m,2H),3.87(s,3H),3.82–3.52(m,18H),3.43(s,2H),2.91–2. 79(m,1H),2.68(d,J=51.2Hz,7H),2.44(d,J=45.9Hz,1H),2.15–2.05(m,1H),1.81–1.65(m,3H).MS-ESI m / z=946.4[M+1] + .

[0247] Example 19

[0248] 2-(2,6-dioxopiperidin-3-yl)-4-((15-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenylethyl)amino)quinazolin-6-yl)piperidin-1-yl)-15-oxo-3,6,9,12-tetraoxopentadecyl)amino)dihydroindole-1,3-dione

[0249]

[0250] Referring to Example 17, ZZ02036 can be obtained using the same method. MS-ESI m / z = 948.4 [M+1] + .

[0251] Example 20

[0252] 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(3-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)-3,6-dihydropyridin-1(2H)-yl)-3-oxopropoxy)ethoxy)ethyl)aminodihydroindole-1,3-dione

[0253]

[0254] Referring to Example 17, RX70052 can be obtained using the same method. MS-ESI m / z = 858.3 [M+1] + .

[0255] Example 21

[0256] (2S,4R)-1-((S)-2-(2-(4-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-carbonyl)piperidin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0257]

[0258] Step 1: Methyl 1-(2-(tert-butoxy)-2-oxoethyl)piperidine-4-carboxylic acid

[0259]

[0260] Methyl piperidine-4-carboxylate (100 mg, 0.70 mmol) was dissolved in dichloromethane (5 mL), and triethylamine (291 μL, 2.10 mmol) and tert-butyl bromoacetate (163 mg, 0.84 mmol) were added sequentially. The mixture was stirred at room temperature for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give methyl 1-(2-(tert-butoxy)-2-oxoethyl)piperidine-4-carboxylate (150 mg, yield 83%).

[0261] Step 2: 2-(4-(methoxycarbonyl)piperidin-1-yl)acetic acid

[0262]

[0263] Methyl 1-(2-(tert-butoxy)-2-oxoethyl)piperidin-4-carboxylic acid (100 mg, 0.39 mmol) was dissolved in dichloromethane (5 mL), and dioxane hydrogen chloride solution (16 mmol, 4 mL) was added dropwise under ice bath conditions. The mixture was stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure, and the crude 2-(4-(methoxycarbonyl)piperidin-1-yl)acetic acid was used directly in the next reaction step.

[0264] Step 3: Methyl 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl))carbamoyl)pyrrolid-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylic acid ester

[0265]

[0266] Methyl 1-(2-(tert-butoxy)-2-oxoethyl)piperidine-4-carboxylic acid (20 mg, 0.10 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (48 mg, 0.10 mmol), and HATU (45 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (4 mL). N,N-diisopropylethylamine (82 μL, 0.50 mmol) was added under ice bath conditions, and the mixture was stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to give methyl 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl))carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylic acid (46 mg, yield 74%).

[0267] Step 4: 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl))carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylic acid

[0268]

[0269] Methyl 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl))carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidin-4-carboxylic acid (46 mg, 0.07 mmol) was dissolved in a mixed solution of tetrahydrofuran (1 mL), methanol (1 mL), and water (1 mL). Lithium hydroxide (18 mg, 0.73 mmol) was added, and the mixture was stirred at 40 °C for 12 hours. The pH was adjusted to 4–5 with saturated citric acid aqueous solution, and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified by Prep-HPLC and freeze-dried, 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl))carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylic acid (31 mg, yield 69%).

[0270] Step 5: (2S,4R)-1-((S)-2-(2-(4-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-carbonyl)piperidin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0271]

[0272] (R)-N-(1-(3-(3-amino-5-(trifluoromethyl)phenyl)ethyl)-7-methoxy-2-methyl-6-(piperidin-4-yl)quinazolin-4-amine (20 mg, 0.04 mmol), 1-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl))carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)piperidin-4-carboxylic acid (27 mg, 0.04 mmol), and HATU (20 mg, 0.05 mmol) were dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (39 μL, 0.22 mmol) was added under ice bath conditions and the mixture was kept warm and stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC to obtain (2S,4R)-1-((S)-2-(2-(4-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazoline-6-yl)piperidin-1-carbonyl)piperidin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (23 mg, yield 50%).

[0273] 1H NMR (500MHz, Methanol-d4) δ8.95(s,1H),8.31(s,1H),7.45(d,J=8.2Hz,2H),7.42(d,J=8.3Hz,2H),7.13–7.00(m,3H),6.97–6.86(m,1 H),5.78(p,J=6.6Hz,1H),5.04–4.97(m,1H),4.78–4.72(m,1H),4.64(s,1H),4.60–4.54(m,1H),4.45(s,1H),4.27–4.17(m,1H),4.08–4 .01(m,4H),3.94–3.85(m,1H),3.75(dd,J=11.0,3.8Hz,1H),3.71–3.59(m,2H),3.54–3.36(m,3H),3.21–3.04(m,3H),2.79(t,J=12.7Hz ,1H),2.63(s,3H),2.49(s,3H),2.26–2.18(m,1H),2.16–1.89(m,9H),1.70(d,J=7.0Hz,3H),1.51(d,J=7.0Hz,3H),1.07(s,9H).MS-ESI m / z=1055.5[M+1] + .

[0274] Example 22

[0275] (2S,4R)-1-((S)-2-(3-(4-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-carbonyl)piperidin-1-yl)propamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0276] Referring to Example 21, ZZ77122 can be obtained using the same method.

[0277]

[0278] 1H NMR(500MHz,DMSO-d6)δ9.85(s,1H),9.20(s,1H),8.99(s,1H),8.39(d,J=7.9Hz,1H), 8.32(d,J=3.3Hz,1H),8.27(d,J=9.2Hz,1H),7.45–7.41(m,2H),7.38(d,J=8.4Hz,2H) ,7.10(s,1H),6.86(s,1H),6.84(s,1H),6.75(d,J=2.1Hz,1H),5.79–5.66(m,1H),4.9 2(p,J=7.0Hz,1H),4.63(d,J=13.2Hz,1H),4.54(d,J=9.3Hz,1H),4.40(t,J=8.2Hz,1H ),4.29(s,1H),4.12(d,J=13.6Hz,1H),4.00(s,4H),3.30–3.25(m,4H),3.18(d,J=17. 8Hz,2H),2.96(s,4H),2.79–2.62(m,2H),2.59(d,J=2.6Hz,3H),2.45(s,2H),2.41–2. 31(m,1H),2.02(t,J=10.2Hz,1H),1.89(d,J=16.9Hz,3H),1.84–1.72(m,4H),1.62(dd ,J=7.1,2.7Hz,3H),1.49(d,J=7.1Hz,1H),1.37(d,J=6.9Hz,3H),0.95(s,9H).MS-ESI m / z=1069.5[M+1] + .

[0279] Example 23

[0280] (2S,4R)-1-((S)-2-((1R,4S)-4-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl]ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidine-1-carbonyl)cyclohexane-1-carboxamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0281] Referring to Example 21, ZZ77112 can be obtained using the same method.

[0282]

[0283] 1H NMR(500MHz,DMSO-d6)δ9.88(d,J=7.8Hz,1H),9.00(s,1H),8.41(d,J=7.5Hz ,1H),8.34(s,1H),7.72(s,1H),7.45(d,J=8.3Hz,2H),7.39(d,J=8.3Hz,2H) ,7.08(s,1H),6.88(s,1H),6.86(s,1H),6.77(d,J=2.1Hz,1H),5.78–5.69(m ,1H),4.93(t,J=7.3Hz,1H),4.66(d,J=12.7Hz,1H),4.51(d,J=9.4Hz,1H),4. 42(t,J=8.1Hz,1H),4.29(s,1H),4.21–4.11(m,1H),4.01(s,3H),3.67–3.55 (m,4H),3.17(d,J=6.6Hz,4H),2.60(s,3H),2.47(s,3H),2.01(q,J=9.0,8.1H z,1H),1.89(s,1H),1.80(q,J=8.2Hz,2H),1.68(s,4H),1.64(d,J=7.1Hz,3H ),1.58–1.41(m,2H),1.39(d,J=7.0Hz,3H),1.25(s,4H),0.94(s,9H).MS-ESI m / z = 1040.5[M+1] + .

[0284] Example 24

[0285] (2S,4R)-1-((S)-2-(4-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)butyramido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0286]

[0287] Step 1: (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline-7-oxy)butyrate

[0288]

[0289] (R)-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-hydroxy-2-methylquinazolin-6-yl)piperidin-1-yl)ethyl-1-one (50 mg, 0.10 mmol) and potassium carbonate (28 mg, 0.20 mmol) were dissolved in acetonitrile (4 mL), and methyl 4-bromobutyrate (28 mg, 0.15 mmol) was added. The mixture was heated to 70 °C and stirred for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to obtain (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline-7-oxy)butyrate (47 mg, yield 78%).

[0290] Step 2: (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)butyric acid

[0291]

[0292] Methyl (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline-7-oxy)butyrate (40 mg, 0.07 mmol) was dissolved in a mixed solution of tetrahydrofuran (1 mL), methanol (1 mL), and water (1 mL). Lithium hydroxide (16 mg, 0.68 mmol) was added, and the mixture was stirred at 40 °C for 12 hours. The pH was adjusted to 4–5 with saturated citric acid aqueous solution, and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Crude (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline-7-yl)oxy)butyrate was used directly in the next reaction step.

[0293] Step 3: (2S,4R)-1-((S)-2-(4-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)butyramido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0294]

[0295] Dissolve (R)-4-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline-7-yl)oxy)butyric acid (30 mg, 0.05 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (25 mg, 0.05 mmol), and HATU (24 mg, 0.06 mmol) in N,N-dimethylformamide (3 mL). Add N,N-diisopropylethylamine (43 μL, 0.26 mmol) under ice bath conditions and keep the mixture warm and stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to give (2S,4R)-1-((S)-2-(4-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)butyramido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (30 mg, yield 57%).

[0296] 1H NMR(500MHz,DMSO-d6)δ9.84(d,J=8.0Hz,1H),8.99(s,1H),8.38(d,J=7.8Hz,1H),8.32 (d,J=3.9Hz,1H),7.87(d,J=9.2Hz,1H),7.43(d,J=8.2Hz,2H),7.37(d,J=8.1Hz,2H),7 .03(s,1H),6.86(s,1H),6.85(s,1H),6.75(s,1H),5.72(p,J=7.7,7.1Hz,1H),4.91(p, J=6.8Hz,1H),4.63(d,J=12.8Hz,1H),4.52(d,J=9.3Hz,1H),4.40(t,J=8.2Hz,1H),4.28 (s,1H),4.17(t,J=6.4Hz,2H),3.99(d,J=13.4Hz,1H),3.61–3.57(m,3H),3.27–3.17(m ,4H),2.68–2.61(m,2H),2.58(s,3H),2.45(s,3H),2.34–2.28(m,1H),2.20–2.12(m,1H) ,2.04(d,J=3.4Hz,3H),2.00(d,J=8.2Hz,1H),1.89–1.82(m,3H),1.81–1.74(m,2H),1. 62(dd,J=7.0,2.0Hz,3H),1.50–1.43(m,2H),1.37(d,J=6.9Hz,3H),0.93(s,9H).MS-ESI m / z=1000.5[M+1] + .

[0297] Example 25

[0298] (2S,4R)-1-((S)-2-(6-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)hexaamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0299] Referring to Example 24, ZZ24064 can be obtained using the same method.

[0300]

[0301] 1H NMR (500MHz, DMSO-d6) δ9.84(d,J=8.1Hz,1H),8.99(s,1H),8.38(d,J=7.8Hz,1H),8.32(d ,J=3.9Hz,1H),7.87(d,J=9.2Hz,1H),7.43(d,J=8.2Hz,2H),7.37(d,J=8.1Hz,2H),7.03( s,1H),6.86(s,1H),6.85(s,1H),6.75(s,1H),5.72(t,J=7.3Hz,1H),4.91(p,J=7.1Hz,1H ),4.63(d,J=12.7Hz,1H),4.52(d,J=9.3Hz,1H),4.40(t,J=8.2Hz,1H),4.28(s,1H),4.17( t,J=6.4Hz,2H),3.99(d,J=13.4Hz,1H),3.66–3.55(m,2H),3.22(t,J=14.2Hz,2H),2.69– 2.60(m,1H),2.58(s,3H),2.45(s,3H),2.34–2.29(m,1H),2.21–2.12(m,1H),2.04(d,J=3 .4Hz,3H),1.85(d,J=6.5Hz,2H),1.78(dt,J=13.0,5.5Hz,1H),1.62(dd,J=7.0,2.0Hz,5H ),1.45(t,J=8.3Hz,2H),1.37(d,J=6.9Hz,3H),1.23(d,J=3.8Hz,2H),0.93(s,9H).MS-ESI m / z=1028.5[M+1] + .

[0302] Example 26

[0303] (2S,4R)-1-((S)-2-(8-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)octadecylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0304] Referring to Example 24, ZZ24065 can be obtained using the same method.

[0305]

[0306] 1H NMR(500MHz,DMSO-d6)δ9.84(d,J=7.9Hz,1H),8.99(s,1H),8.39(d,J=7.8Hz,1H), 8.32(d,J=3.4Hz,1H),7.82(d,J=9.3Hz,1H),7.43(d,J=8.3Hz,2H),7.37(d,J=8.3H z,2H),7.05(d,J=4.8Hz,1H),6.86(s,1H),6.85(s,1H),6.76(s,1H),5.78–5.68(m ,1H),4.91(p,J=7.0Hz,1H),4.62(d,J=12.8Hz,1H),4.52(d,J=9.3Hz,1H),4.41(t, J=8.1Hz,1H),4.27(s,1H),4.17(t,J=6.4Hz,2H),3.98(t,J=11.2Hz,1H),3.18(q, J=14.1Hz,2H),2.66–2.61(m,1H),2.58(s,3H),2.45(s,3H),2.33–2.24(m,1H),2.1 5–2.06(m,1H),2.06–1.97(m,4H),1.88–1.75(m,4H),1.62(dd,J=7.2,1.8Hz,3H),1 .45(d,J=7.7Hz,3H),1.37(d,J=7.0Hz,4H),1.32–1.22(m,2H),0.92(s,9H).MS-ESI m / z=1056.5[M+1] + .

[0307] Example 27

[0308] (2S,4R)-1-((S)-2-(10-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)decamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0309] Referring to Example 24, ZZ24066 can be obtained using the same method.

[0310]

[0311] 1H NMR(500MHz,DMSO-d6)δ9.88–9.81(m,1H),8.99(s,1H),8.39(d,J=7.8Hz,1H),8.32(d, J=3.5Hz,1H),7.81(d,J=9.4Hz,1H),7.43(d,J=8.3Hz,2H),7.37(d,J=8.2Hz,2H),7.04( s,1H),6.86(s,1H),6.85(s,1H),6.76(d,J=2.2Hz,1H),5.72(t,J=7.3Hz,1H),4.91(p,J =7.2Hz,1H),4.62(d,J=12.7Hz,1H),4.51(d,J=9.4Hz,1H),4.40(t,J=8.1Hz,1H),4.27( s,1H),4.17(t,J=6.3Hz,2H),3.98(d,J=13.5Hz,1H),3.26–3.11(m,2H),2.65–2.60(m, 1H),2.58(d,J=1.1Hz,3H),2.45(s,3H),2.36(p,J=1.9Hz,1H),2.26(dt,J=14.9,7.6Hz, 1H),2.13–2.06(m,1H),2.05–1.98(m,4H),1.88–1.75(m,4H),1.62(dd,J=7.0,1.8Hz,3H ),1.52–1.43(m,4H),1.37(d,J=7.0Hz,3H),1.26(d,J=23.3Hz,9H),0.92(s,9H).MS-ESI m / z=1084.5[M+1] + .

[0312] Example 28

[0313] (2S,4R)-1-((S)-2-(2-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0314]

[0315] Referring to Example 24, ZZ24101 can be obtained using the same method. MS-ESI m / z = 972.4 [M+1] + .

[0316] Example 29

[0317] (2S,4R)-1-((S)-2-(5-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0318]

[0319] Referring to Example 24, ZZ24102 can be obtained using the same method. MS-ESI m / z = 1014.5 [M+1] + .

[0320] Example 30

[0321] 4-((5-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0322]

[0323] Step 1: 5-((tert-butoxycarbonyl)amino)4-methylbenzenesulfonate pentyl ester

[0324]

[0325] 100 mg (0.49 mmol) of tert-butyl (5-hydroxypentyl)carbamate was dissolved in 5 mL of dichloromethane. Triethylamine (205 μL, 1.48 mmol) and p-methylsulfonyl chloride (103 mg, 0.54 mmol) were added sequentially under ice bath conditions, and the mixture was stirred for 12 hours. The mixture was then extracted with water and dichloromethane. The organic phase was separated, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 6 / 1) to give 150 mg (85% yield) of pentyl 5-((tert-butoxycarbonyl)amino)4-methylbenzenesulfonate.

[0326] Step 2: (R)-(5-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentyl)tert-butyl carbamate

[0327]

[0328] (R)-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-hydroxy-2-methylquinazoline-6-yl)piperidin-1-yl)ethyl-1-one (30 mg, 0.06 mmol), 5-((tert-butoxycarbonyl)amino)4-methylbenzenesulfonate (33 mg, 0.09 mmol), and potassium carbonate (26 mg, 0.18 mmol) were dissolved in N,N-dimethylformamide (4 mL), and the mixture was stirred at 70 °C for 16 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to give (R)-(5-((6-(1-acetylpiperidin-4-yl)-4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentyl)carbamate tert-butyl ester (25 mg, yield 60%).

[0329] Step 3: (R)-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-(5-aminopropoxy)-2-methylquinazolin-6-yl)piperidin-1-yl)ethyl-1-one

[0330]

[0331] (R)-(50 mg, 0.07 mmol) tert-butyl carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise under ice bath conditions. The mixture was kept warm and stirred for 2 hours. The crude product of (R)-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-(5-aminopropoxy)-2-methylquinazoline-6-yl)piperidin-1-yl)ethyl-1-one was used directly in the next step of the reaction.

[0332] Step 4: 4-((5-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0333]

[0334] (R)-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-(5-aminopropoxy)-2-methylquinazolin-6-yl)piperidin-1-yl)ethyl-1-one (30 mg, 0.05 mmol) and 2-(2,6-dioxadipinidin-3-yl)-4-fluoroisoindole-1,3-dione (22 mg, 0.08 mmol) were dissolved in dimethyl sulfoxide, and N,N-diisopropylethylamine (47 μL, 0.26 mmol) was added. The mixture was stirred at 80 °C for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC to obtain 4-((5-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (31 mg, yield 71%).

[0335] 1 H NMR (400MHz, DMSO-d6) δ11.10(s,1H),8.30(s,1H),7.58(t,J=7.8Hz,1H),7.22(s,1H),7.12(d,J=8.5Hz,1H),7.03(s,1H),6.85( d,J=6.5Hz,2H),6.75(s,1H),6.68(s,1H),6.59(t,J=5.6Hz,1H),5.78–5.59(m,2H),5.32(t,J=4.7Hz,2H),5.04(dd,J=12.5,5.4 Hz,1H),4.59(d,J=12.2Hz,1H),4.23–4.15(m,2H),3.91(d,J=13.6Hz,1H),3.24–3.15(m,1H),3.13–3.02(m,1H),2.93–2.79(m,1 H),2.62–2.53(m,6H),2.03–1.96(m,4H),1.93–1.85(m,2H),1.73–1.65(m,2H),1.65–1.52(m,7H),1.45(t,J=7.1Hz,3H).MS-ESI m / z=829.4[M+1] + .

[0336] Example 31

[0337] (2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(3-((6-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-6-oxohexyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide

[0338]

[0339] Step 1: Methyl 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carbamoyl)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carbamoyl)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoate

[0340]

[0341] (2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(3-hydroxy-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (51 mg, 0.10 mmol) and potassium carbonate (40 mg, 0.29 mmol) were dissolved in N,N-dimethylformamide (4 mL), and methyl 6-bromohexanoate (30 mg, 0.14 mmol) was added. The mixture was stirred at 70 °C for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to give methyl 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carbamoyl)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carbamoyl)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoate (40 mg, yield 63%).

[0342] Step 2: 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido))-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoic acid

[0343]

[0344] Methyl 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoate (40 mg, 0.06 mmol) was dissolved in a mixed solution of tetrahydrofuran (2 mL), methanol (2 mL), and water (2 mL), and lithium hydroxide (14 mg, 0.60 mmol) was added. The mixture was stirred at 40°C for 12 hours. The pH was adjusted to 4–5 with saturated citric acid aqueous solution, and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido))-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoic acid was used directly in the next step of the reaction.

[0345] Step 3: (2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(3-((6-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-6-oxohexyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide

[0346]

[0347] (R)-7-methoxy-2-methyl-6-(piperidin-4-yl)-N-(1-(3-(trifluoromethyl)phenyl)ethyl)quinazolin-4-amine (30 mg, 0.07 mmol), 6-(5-((((2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido))-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-2-(4-methylthiazolyl-5-yl)phenoxy)hexanoic acid (44 mg, 0.07 mmol) and HATU (31 mg, 0.08 mmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (56 μL, 0.34 mmol) was added under ice bath conditions. The mixture was kept warm and stirred for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC and freeze-dried to give (2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(3-((6-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-6-oxohexyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, yield 41%).

[0348] 1H NMR (500MHz, DMSO-d6) δ9.90(d,J=7.7Hz,1H),8.96(d,J=4.2Hz,1H),8.50(t,J=6.1Hz,1H),8.31( s,1H),7.81(s,1H),7.77(d,J=7.6Hz,1H),7.65(d,J=7.8Hz,1H),7.60(t,J=7.8Hz,1H),7.39(d,J= 7.7Hz,1H),7.32–7.23(m,1H),7.11(s,1H),6.98(s,1H),6.94(d,J=7.8Hz,1H),5.84(t,J=7.5Hz,1 H),4.64(d,J=13.3Hz,1H),4.58(d,J=9.2Hz,1H),4.51(t,J=8.2Hz,1H),4.34(s,1H),4.32–4.25(m ,1H),4.20(dd,J=16.6,5.7Hz,1H),4.04(q,J=8.0,7.1Hz,3H),3.98(s,3H),3.67–3.58(m,4H),3.2 3(d,J=12.1Hz,1H),3.14(t,J=13.3Hz,1H),2.63(t,J=12.8Hz,1H),2.56(s,3H),2.44(s,3H),2.43 –2.31(m,2H),2.08(dd,J=12.9,7.8Hz,1H),1.97–1.72(m,5H),1.71–1.65(m,3H),1.59(q,J=7.7Hz ,2H),1.49(p,J=6.7Hz,2H),1.34(dt,J=19.3,11.4Hz,2H),1.27–1.17(m,3H),0.94(s,9H).MS-ESI m / z=1073.5[M+1] + .

[0349] Example 32

[0350] (2R,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(3-(((7-(4-(7-methoxy-2-methyl-4-(((R)-1-(3-(trifluoromethyl)phenyl)ethyl)amino)quinazolin-6-yl)piperidin-1-yl)-7-oxohepyl)oxy)-4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide

[0351] Referring to Example 31, RX10041 can be obtained using the same method.

[0352]

[0353] 1 H NMR(500MHz,DMSO-d6)δ9.85(s,1H),8.97(s,1H),8.49(d,J=5.9Hz,1H),8.31(s,1H),7.81 (s,1H),7.77(d,J=7.7Hz,1H),7.63(d,J=16.8Hz,2H),7.40(d,J=7.7Hz,1H),7.28(d,J=9. 2Hz,1H),7.08(s,1H),6.99(d,J=6.0Hz,1H),6.94(d,J=7.9Hz,1H),5.92–5.77(m,1H),5.1 7(s,1H),4.64(d,J=13.7Hz,1H),4.59(d,J=9.1Hz,1H),4.51(t,J=8.4Hz,1H),4.34(s,1H) ,4.32–4.15(m,2H),4.04(s,4H),3.99(s,3H),3.59(d,J=19.3Hz,3H),3.26(d,J=12.8Hz,1 H),3.14(t,J=13.2Hz,1H),2.61(d,J=15.1Hz,2H),2.56(s,3H),2.45(d,J=3.9Hz,3H),2.4 2–2.26(m,1H),2.07(s,1H),1.96–1.80(m,1H),1.75(d,J=9.1Hz,2H),1.68(d,J=7.1Hz,3H ),1.59–1.43(m,4H),1.43–1.32(m,4H),1.22(d,J=12.6Hz,4H),1.01–0.89(m,9H).MS-ESI m / z=1087.5[M+1] + .

[0354] Example 33

[0355] (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0356]

[0357]

[0358] Step 1: tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-methoxy-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0359]

[0360] tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (50 mg, 0.09 mmol), methyl 10-bromodecanoate (35 mg, 0.13 mmol), and potassium carbonate (36 mg, 0.26 mmol) were dissolved in N,N-dimethylformamide (3 mL) and stirred at 70 °C for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to give tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-methoxy-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (36 mg, yield 54%).

[0361] Step 2: 10-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoic acid

[0362]

[0363] 36 mg (0.05 mmol) of tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-methoxy-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was dissolved in a mixture of tetrahydrofuran (2 mL), methanol (2 mL), and water (2 mL). Lithium hydroxide (12 mg, 0.48 mmol) was added, and the mixture was stirred at 40 °C for 12 hours. The pH was adjusted to 4–5 with saturated citric acid aqueous solution, and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude 10-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoic acid obtained was used directly in the next step of the reaction.

[0364] Step 3: Tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0365]

[0366] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (25 mg, 0.05 mmol), 10-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)- 6-Chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoic acid (38 mg, 0.05 mmol) and HATU (24 mg, 0.06 mmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (43 μL, 0.26 mmol) was added under ice bath conditions. The mixture was stirred and kept at this temperature for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, then washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-p-TLC (dichloromethane / methanol = 20 / 1) to give tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (40 mg, yield 66%).

[0367] Step 4: (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0368]

[0369] 40 mg (0.03 mmol) of tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(10-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-10-oxododecyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was dissolved in dichloromethane (3 mL) under ice bath conditions. Add 1 mL of trifluoroacetic acid dropwise, keep warm and stir for 2 hours, and concentrate under reduced pressure. Purify by Prep-HPLC and freeze-dry to obtain (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (24 mg, yield 50%).

[0370] 1H NMR(500MHz,DMSO-d6)δ9.61(s,1H),8.99(s,1H),8.57(s,2H),8.39(d,J=7.8Hz,1H), 7.81(d,J=9.3Hz,1H),7.46–7.40(m,2H),7.40–7.35(m,2H),6.87(d,J=1.8Hz,1H),6. 81(d,J=6.8Hz,2H),6.56(d,J=1.8Hz,1H),5.11(s,2H),4.90(h,J=7.1Hz,1H),4.52(d ,J=9.3Hz,1H),4.41(dd,J=10.2,6.0Hz,3H),4.28(s,1H),4.24(s,4H),4.13(t,J=6.2H z,4H),3.63–3.54(m,4H),3.16(s,2H),3.08(t,J=12.4Hz,2H),2.45(s,3H),2.25(dq, J=15.4,7.7Hz,1H),2.16(d,J=2.0Hz,6H),2.10(ddd,J=14.5,8.3,6.3Hz,1H),2.01(dd ,J=12.7,7.7Hz,1H),1.78(ddt,J=13.0,9.1,4.6Hz,1H),1.68(s,2H),1.50(dq,J=26. 8,6.7,6.0Hz,2H),1.37(d,J=7.0Hz,3H),1.29(d,J=17.3Hz,12H),0.93(s,9H).MS-ESI m / z=1065.5[M+1] + .

[0371] Example 34

[0372] (2S,4R)-1-((S)-2-(6-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-ylhexamethylene)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0373] Referring to Example 33, ZZ24010 can be obtained using the same method.

[0374]

[0375] 1H NMR(500MHz,DMSO-d6)δ8.99(s,1H),8.60(s,2H),8.39(d,J=7.8Hz,1H),7.86 (d,J=9.3Hz,1H),7.43(d,J=7.9Hz,2H),7.37(d,J=8.2Hz,2H),6.88(d,J=1.8H z,1H),6.81(d,J=6.8Hz,2H),6.56(d,J=1.9Hz,1H),5.11(s,2H),4.91(p,J=7 .2Hz,1H),4.53(d,J=9.4Hz,1H),4.45–4.38(m,3H),4.29(s,1H),4.25(s,4H), 4.13(t,J=6.2Hz,6H),3.65–3.56(m,4H),3.25–3.16(m,2H),3.11–3.01(m,4H ),2.45(s,3H),2.34–2.27(m,1H),2.16(d,J=2.0Hz,6H),2.01(q,J=9.4,7.6Hz ,1H),1.79(ddd,J=13.0,8.7,4.6Hz,1H),1.74–1.64(m,2H),1.55(tq,J=14.0 ,7.0Hz,2H),1.37(d,J=7.0Hz,3H),1.30(q,J=7.4Hz,2H),0.94(s,9H).MS-ESI m / z = 1009.5 [M+1] + .

[0376] Example 35

[0377] (2S,4R)-1-((S)-2-(11-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptadi-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)undecanoamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0378] Referring to Example 33, ZZ24048 can be obtained using the same method.

[0379]

[0380] 1H NMR(500MHz,DMSO-d6)δ8.99(s,1H),8.56(s,2H),8.38(d,J=7.8Hz,1H),7.80 (d,J=9.3Hz,1H),7.47–7.41(m,2H),7.37(d,J=8.3Hz,2H),6.87(d,J=1.8Hz, 1H),6.81(d,J=6.8Hz,2H),6.56(d,J=1.8Hz,1H),5.10(s,2H),4.91(p,J=7.1 Hz,1H),4.52(d,J=9.4Hz,1H),4.45–4.38(m,3H),4.28(s,1H),4.24(s,4H),4 .13(t,J=6.1Hz,4H),3.23–3.12(m,4H),3.07(t,J=12.4Hz,2H),2.45(s,3H), 2.26(dt,J=14.9,7.7Hz,1H),2.16(d,J=2.0Hz,6H),2.13–2.06(m,1H),2.01( t,J=10.4Hz,1H),1.79(ddd,J=13.0,8.7,4.7Hz,1H),1.73–1.63(m,2H),1.55 –1.42(m,2H),1.37(d,J=7.0Hz,3H),1.33–1.20(m,12H),0.93(s,9H).MS-ESI m / z = 1079.5 [M+1] + .

[0381] Example 36

[0382] (2S,4R)-1-((S)-2-(12-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)dodecanoamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0383] Referring to Example 33, ZZ24049 can be obtained using the same method.

[0384]

[0385] 1H NMR (500MHz, DMSO-d6) δ8.99 (s, 1H), 8.62 (s, 2H), 8.38 (d, J = 7.7Hz, 1H), 7.80 ( d,J=9.3Hz,1H),7.46–7.40(m,2H),7.40–7.36(m,2H),6.87(d,J=1.8Hz,1H),6. 81(d,J=6.7Hz,2H),6.56(d,J=1.9Hz,1H),5.10(s,2H),4.91(p,J=7.3Hz,1H), 4.52(d,J=9.2Hz,1H),4.44–4.38(m,3H),4.27(s,1H),4.25(s,4H),4.13(d,J=6 .2Hz,4H),3.61(dd,J=10.7,4.3Hz,4H),3.23–3.12(m,4H),3.08(t,J=12.5Hz, 2H),2.45(s,3H),2.25(dt,J=14.9,7.5Hz,1H),2.16(d,J=2.1Hz,6H),2.14–2.0 5(m,1H),2.01(t,J=10.1Hz,1H),1.82–1.74(m,1H),1.68(s,2H),1.47(dt,J=1 6.6,8.3Hz,2H),1.37(d,J=7.0Hz,3H),1.34–1.17(m,14H),0.93(s,9H).MS-ESI m / z = 1093.6 [M+1] + .

[0386] Example 37

[0387] (2S,4R)-1-((S)-2-(8-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptadi-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yloctadecyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0388] Referring to Example 33, ZZ77153 can be obtained using the same method.

[0389]

[0390] 1H NMR(500MHz,DMSO-d6)δ9.70(s,1H),9.00(s,1H),8.63(s,2H),8.42(d,J=7 .8Hz,1H),7.85(d,J=9.3Hz,1H),7.43(d,J=8.1Hz,2H),7.37(d,J=8.2Hz,2H ),6.88(s,1H),6.81(d,J=6.8Hz,2H),6.57(s,1H),5.11(s,2H),4.90(p,J=6 .9Hz,1H),4.52(d,J=9.3Hz,1H),4.43–4.34(m,3H),4.25(s,5H),4.13(t,J= 6.2Hz,4H),3.23–3.11(m,4H),3.07(t,J=12.2Hz,2H),2.45(s,3H),2.27(d t,J=14.8,7.7Hz,1H),2.20–2.07(m,7H),2.00(ddd,J=16.9,12.5,6.7Hz,1H ),1.78(ddd,J=13.3,8.9,4.8Hz,1H),1.68(q,J=7.9Hz,2H),1.49(dq,J=22. 5,7.0Hz,2H),1.37(d,J=6.9Hz,3H),1.32–1.19(m,8H),0.93(s,9H).MS-ESI m / z = 1037.5 [M+1] + .

[0391] Example 38

[0392] (2S,4R)-1-((S)-2-(7-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)heptamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0393] Referring to Example 33, ZC79018 can be obtained using the same method.

[0394]

[0395] 1H NMR(500MHz,DMSO-d6):8.99(s,1H),8.37(d,J=7.8Hz,1H),7.81(d,J=9.4Hz,1H) ,7.48–7.31(m,6H),7.06–7.01(m,1H),6.98(d,J=1.6Hz,1H),6.57(d,J=1.5Hz,1H ),5.21(s,2H),4.92(dd,J=14.3,7.1Hz),4.53(d,J=9.4Hz,1H),4.46–4.37(m,3H) ,4.32–4.27(m,1H),4.25(s,4H),4.13(t,J=6.0Hz,4H),3.97–3.31(m,6H,covered by the peakof water),3.25–3.02(m,6H),2.46(s,3H),2.32–2.08(m,2H),2.06–1.98(m,1H),1.84–1.76(m,1H),1 .73–1.61(m,2H),1.60–1.44(m,2H),1.38(d,J=7.0Hz,3H),1.35–1.26(m,4H),0.95(s,9H).MS-ESI m / z=1029.4[M+1] + .

[0396] Example 39

[0397] (2S,4R)-1-((S)-2-(9-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)nonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0398] Referring to Example 33, ZC79022 can be obtained using the same method.

[0399]

[0400] 1H NMR (500MHz, DMSO-d6): 8.99 (s, 1H), 8.35 (t, J = 9.0Hz, 1H), 7.78 (t, J = 8.8Hz, 1H), 7.50–7.31 (m, 6H), 7.02 (d, J = 11.9Hz, 1H), 6. 97(d,J=6.2Hz,1H),6.56(d,J=17.2Hz,1H),5.20(s,2H),4.95–4.87(m,1H),4.53(d,J=9.4Hz,1H),4.45–4.38(m,3H),4.31–4.26 (m,1H),4.24(s,4H),4.13(t,J=6.0Hz,4H),3.65–3.55(m,6H),3.24–3.03(m,6H),2.45(s,3H),2.32–2.07(m,2H),2.05–1.97(m, 1H),1.85–1.76(m,1H),1.72–1.62(m,2H),1.60–1.41(m,2H),1.38(d,J=6.9Hz,3H),1.25(d,J=47.3Hz,8H),0.94(s,9H).MS-ESI m / z=1057.5[M+1] + .

[0401] Example 40

[0402] (2S,4R)-1-((S)-2-(8-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)octadecylamine)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0403] Referring to Example 33, ZC79024 can be obtained using the same method.

[0404]

[0405] 1H NMR (500MHz, DMSO-d6): δ8.99(s,1H),8.37(d,J=7.8Hz,1H),7.80(d,J=9.3Hz,1H),7.52–7.30(m,6H),7.07–7.01(m,1H),6 .98(d,J=1.5Hz,1H),6.58(t,J=3.5Hz,1H),5.22(s,2H),4.96–4.85(m,1H),4.53(d,J=9.4Hz,1H),4.42(t,J=8.1Hz,3H),4. 32–4.21(m,5H),4.13(t,J=6.1Hz,4H),3.66–3.54(m,4H),3.00–3.25(m,8H),2.46(s,3H),2.33–2.08(m,2H),2.07–1.98(m, 1H),1.84–1.76(m,1H),1.74–1.62(m,2H),1.59–1.43(m,2H),1.38(d,J=7.0Hz,3H),1.35–1.22(m,6H),0.94(s,9H).MS-ESI m / z=1043.5[M+1] + .

[0406] Example 41

[0407] (2S,4R)-1-((S)-2-(5-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)pentamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0408] Referring to Example 33, ZC79054 can be obtained using the same method.

[0409]

[0410] 1H NMR (500MHz, DMSO-d6): δ9.00(s,1H),8.39(d,J=7.8Hz,1H),7.93(d,J=9.2Hz,1H),7.48–7.32(m,6H),7.03(ddd,J=8.4,4.5,2.2Hz,1 H),6.99(d,J=1.6Hz,1H),6.58(d,J=1.5Hz,1H),5.22(s,2H),4.90(td,J=14.2,7.0Hz,1H),4.53(d,J=9.3Hz,1H),4.45–4.37(m,3H), 4.32–4.22(m,5H),4.13(t,J=6.0Hz,4H),3.66–3.53(m,4H),3.28–3.00(m,6H),2.55–2.47(m,2H),2.45(s,3H),2.40–2.17(m,2H),2. 08–1.98(m,1H),1.79(ddd,J=12.9,8.7,4.6Hz,1H),1.73–1.62(m,2H),1.61–1.51(m,2H),1.36(d,J=7.0Hz,3H),0.95(s,9H).MS-ESI m / z=1001.4[M+1] + .

[0411] Example 42

[0412] (2S,4R)-1-((S)-2-(6-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)hexaamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0413] Referring to Example 33, ZC79055 can be obtained using the same method.

[0414]

[0415] 1H NMR (500MHz, DMSO-d6): δ9.00(s,1H),8.40(d,J=7.8Hz,1H),7.87(d,J=9.3Hz,1H),7.48–7.32(m,6H,),7.03(ddd,J=8.5,4.5,2.1Hz,1H), 6.99(d,J=1.6Hz,1H),6.58(d,J=1.5Hz,1H),5.22(s,2H),4.98–4.86(m,1H),4.53(d,J=9.4Hz,1H),4.46–4.37(m,3H),4.32–4.18(m,5H), 4.13(t,J=6.0Hz,4H),3.66–3.54(m,4H),3.26–3.02(m,6H),2.55–2.47(m,2H),2.45(s,3H),2.35–2.12(m,2H),2.08–1.98(m,1H),1.79(d dd,J=12.9,8.7,4.6Hz,1H),1.75–1.63(m,2H),1.62–1.45(m,2H),1.37(d,J=7.0Hz,3H),1.30(dd,J=14.7,7.3Hz,2H),0.94(s,9H).MS-ESI m / z=1015.4[M+1] + .

[0416] Example 43

[0417] (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0418] Referring to Example 33, ZC79058 can be obtained using the same method.

[0419]

[0420] 1H NMR(500MHz, DMSO-d6):8.99(s,1H),8.40(d,J=7.8Hz,1H),7.82(d,J=9.3Hz,1H),7.48–7.31(m,6H),7.03(ddd,J=8.4,4.5,2.1Hz,1H),6 .99(d,J=1.6Hz,1H),6.58(d,J=1.5Hz,1H),5.22(s,2H),4.90(ddd,J=21.5,14.3,7.1Hz,1H),4.52(d,J=9.4Hz,1H),4.45–4.35(m,3H),4 .31–4.19(m,5H),4.13(t,J=6.0Hz,4H),3.69–3.53(m,4H),3.24–3.00(m,6H),2.54–2.47(m,2H),2.45(s,3H),2.31–2.06(m,2H),2.05–1 .97(m,1H),1.83–1.74(m,1H),1.73–1.61(m,2H),1.57–1.40(m,2H),1.37(d,J=7.0Hz,3H),1.28(d,J=16.7Hz,10H),0.93(s,9H).MS-ESI m / z=1071.5[M+1] + .

[0421] Example 44

[0422] (2S,4R)-1-((S)-2-(7-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptyl-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)heptamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0423] Referring to Example 33, ZC79069 can be obtained using the same method.

[0424]

[0425] 1H NMR (500MHz, DMSO) δ9.00 (s, 1H), 8.40 (d, J = 7.8Hz, 1H), 7.85 (d, J = 9.3Hz, 1H), 7.47–7. 35(m,4H,-ArH),6.89(d,J=1.5Hz,1H),6.81(d,J=6.8Hz,2H),6.57(d,J=1.2Hz,1H),5. 11(s,2H),4.96–4.85(m,1H),4.53(d,J=9.4Hz,1H),4.44–4.34(m,3H),4.31–4.22(m,5 H),4.13(t,J=5.9Hz,4H),3.68–3.53(m,4H),3.25–3.00(m,6H),2.53–2.48(2H,partly covered by the peak of solvent),2.45(s,3H),2.33–2.23(m,1H),2.19–2.09(m,7H),2.06–1.98(m,1H),1.83–1.74(m,1 H),1.67(s,2H),1.59–1.43(m,2H),1.37(d,J=7.0Hz,3H),1.34–1.22(m,4H),0.94(s,9H).MS-ESI m / z=1023.5[M+1] + .

[0426] Example 45

[0427] (2S,4R)-1-((S)-2-(9-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptadi-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)nonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0428] Referring to Example 33, ZC79070 can be obtained using the same method.

[0429]

[0430] 1H NMR(500MHz,DMSO-d6):8.99(s,1H),8.41(d,J=7.8Hz,1H),7.83(d,J=9.3Hz,1H), 7.47–7.35(m,4H),6.89(d,J=1.4Hz,1H),6.81(d,J=6.8Hz,2H),6.57(s,1H),5.11( s,2H),4.96–4.86(m,1H),4.52(d,J=9.4Hz,1H),4.45–4.33(m,3H),4.31–4.23(m,5 H),4.13(t,J=6.0Hz),3.66–3.54(m,4H),3.26–3.02(m,6H),2.53–2.48(2H,partly covered by the peak of solvent),2.45(s,3H),2.31–2.22(m,1H),2.19–2.07(m,7H),2.06–1.97(m,1H),1.83–1.74(m ,1H),1.68(s,2H),1.59–1.41(m,2H),1.37(d,J=7.0Hz,3H),1.30(s,8H),0.93(s,9H).MS-ESI m / z=1051.5[M+1] + .

[0431] Example 46

[0432] (2S,4R)-1-((S)-2-(8-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)octamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0433]

[0434]

[0435] Step 1: 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-methoxy-8-oxooctyl)-3,5-dimethyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester

[0436]

[0437] 6-(6-chloro-4-(3,5-dimethyl-1H-pyrazol-4-yl)-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3,3]heptane-2-carboxylic acid tert-butyl ester (50 mg, 0.09 mmol) and potassium carbonate (36 mg, 0.26 mmol) were dissolved in N,N-dimethylformamide, and methyl 8-bromooctanoate (31 mg, 0.13 mmol) was added. The mixture was stirred at 70 °C for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-TLC (dichloromethane / methanol = 20 / 1) to give 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-methoxy-8-oxoctyl)-3,5-dimethyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (48 mg, yield 76%).

[0438] Step 2: 8-(4-(2-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]hept-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)octanoic acid

[0439]

[0440] Tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-methoxy-8-oxooctyl)-3,5-dimethyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was dissolved in a mixed solution of tetrahydrofuran (2 mL), methanol (2 mL), and water (2 mL). Lithium hydroxide was added, and the mixture was stirred at 40 °C for 12 hours. The pH was adjusted to 4–5 with saturated citric acid aqueous solution, and the mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude 8-(4-(2-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]hept-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)octanoic acid was used directly in the next step of the reaction.

[0441] Step 3: 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-8-oxoctyl)-3,5-dimethyl-1H-pyrazole-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester

[0442]

[0443] 8-(4-(2-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]hept-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)octanoic acid (50 mg, 0.07 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethyl (Butyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (33 mg, 0.07 mmol) and HATU (32 mg, 0.08 mmol) were dissolved in N,N-dimethylformamide. N,N-diisopropylethylamine (58 μL, 0.35 mmol) was added under ice bath conditions, and the mixture was stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Prep-TLC (dichloromethane / methanol = 10 / 1) to give 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-8-oxoctyl)-3,5-dimethyl-1H-pyrazole-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (40 mg, yield 50%).

[0444] Step 4: (2S,4R)-1-((S)-2-(8-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)octamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0445]

[0446] 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(1-(8-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-8-oxoctyl)-3,5-dimethyl-1H-pyrazole-4-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (40 mg, 0.03 mmol) was dissolved in dichloromethane (2 mL), and added dropwise under ice bath conditions. Add 0.5 mL of trifluoroacetic acid and stir for 2 hours. After concentration under reduced pressure, purify by Prep-HPLC and freeze-dry to obtain (2S,4R)-1-((S)-2-(8-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazole-1-yl)octamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (26 mg, yield 65%).

[0447] 1H NMR (500MHz, DMSO-d6) δ8.99(s,1H),8.57(s,2H),8.38(d,J=7.8Hz,1H),7.80(d,J=9.3Hz,1H),7.43(d,J=8.0Hz,2H),7.37(d,J=8.1Hz,2 H),7.31–7.25(m,1H),6.98(s,1H),6.94(d,J=6.8Hz,2H),5.20(s,2H),4.91(p,J=7.2Hz,1H),4.52(d,J=9.3Hz,1H),4.41(t,J=8.1Hz,2H ),4.36(s,4H),4.28(s,2H),4.13(t,J=6.2Hz,6H),2.45(s,3H),2.27(dt,J=14.6,7.6Hz,1H),2.19(d,J=2.0Hz,6H),2.16(s,3H),2.15–2 .11(m,1H),2.09(s,3H),2.04–1.98(m,1H),1.83–1.70(m,3H),1.56–1.43(m,3H),1.37(d,J=7.0Hz,3H),1.31(s,6H),0.93(s,9H).MS-ESI m / z=1047.4[M+1] + .

[0448] Example 47

[0449] (2S,4R)-1-((S)-2-(9-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro]]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)nonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0450]

[0451] Referring to Example 46, ZZ77116 can be obtained using the same method. MS-ESI m / z = 1061.5 [M+1] + .

[0452] Example 48

[0453] (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro][3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)-3,5-dimethyl-1H-pyrazol-1-yl)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide)

[0454]

[0455] Referring to Example 46, ZZ77117 can be obtained using the same method. MS-ESI m / z = 1074.5 [M+1] + .

[0456] Example 49

[0457] (2S,4R)-1-((S)-2-(2-(4-((4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0458]

[0459] Step 1: Methyl 2-(4-(hydroxymethyl)piperidin-1-yl)acetate

[0460]

[0461] Piperidin-4-ylmethanol (300 mg, 2.60 mmol) was dissolved in dichloromethane (20 mL), and triethylamine (1.1 mL, 7.81 mmol) and ethyl bromoacetate (358 mg, 2.34 mmol) were added sequentially under ice bath conditions. The mixture was stirred for 12 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give methyl 2-(4-(hydroxymethyl)piperidin-1-yl)acetate (450 mg, yield 92%).

[0462] Step 2: Methyl 2-(4-formylpiperidin-1-yl)acetate

[0463]

[0464] Dimethyl sulfoxide (250 mg, 3.20 mmol) was dissolved in dry dichloromethane (20 mL) and cooled to -78 °C. Oxaloyl chloride (135 μL, 1.60 mmol) was slowly added dropwise, and the mixture was stirred for 30 min. A solution of methyl 2-(4-(hydroxymethyl)piperidin-1-yl)acetate (150 mg, 0.08 mmol) in dichloromethane was then slowly added dropwise, and the mixture was stirred for 30 min. Triethylamine (1.11 mL, 8.01 mmol) was added, and the mixture was stirred for 30 min, then cooled to room temperature and stirred for another 30 min. Water was added, the organic phase was separated, and washed with a saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to obtain methyl 2-(4-formylpiperidin-1-yl)acetate, which was used directly in the next reaction.

[0465] Step 3: 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(((1-(2-methoxy-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester

[0466]

[0467] Methyl 2-(4-formylpiperidin-1-yl)acetate (33 mg, 0.18 mmol) and 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (50 mg, 0.09 mol) were dissolved in dichloromethane, and sodium triacetylborohydride (56 mg, 0.26 mmol) was added under ice bath conditions. The mixture was stirred at room temperature for 12 hours. Water was added, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by Prep-TLC to give 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(((1-(2-methoxy-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (45 mg, yield 69%).

[0468] Step 4: 2-(4-((4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetic acid

[0469]

[0470] 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(((1-(2-methoxy-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (45 mg, 0.06 mmol) was dissolved in a mixed solution of tetrahydrofuran (2 mL), methanol (2 mL), and water (2 mL), and lithium hydroxide (15 mg, 0.61 mmol) was added. Stirred at 40°C for 12 hours. Adjust the pH to 4-5 with saturated citric acid aqueous solution, extract with ethyl acetate and water, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and the crude product of 2-(4-((4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetic acid was used directly for the next step of the reaction.

[0471] Step 5: 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-((1-(2-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutyr-2-yl)amino)-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester

[0472]

[0473] 2-(4-((4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzimidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetic acid (40 mg, 0.06 mmol), (2S,4R)-1-((S)-2-amino-3,3-di Methylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (27 mg, 0.06 mmol) and HATU (25 mg, 0.07 mmol) were dissolved in N,N-dimethylformamide (4 mL). N,N-diisopropylethylamine (46 μL, 0.28 mmol) was added under ice bath conditions, and the mixture was stirred for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was separated and washed three times with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative TLC (dichloromethane / methanol = 10 / 1) to obtain 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-((1-(2-((((S)-1-((2S,4R)-4-hydroxy) -2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutanol-2-yl)amino)-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (52 mg, yield 82%).

[0474] Step 6: (2S,4R)-1-((S)-2-(2-(4-((4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0475]

[0476] 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-((1-(2-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutyr-2-yl)amino)-2-oxoethyl)piperidin-4-yl)methyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (52 mg, 0.05 mmol) was dissolved in dichloromethane (4 mL) under ice bath conditions. Trifluoroacetic acid was added dropwise to the mixture, and after stirring at a constant temperature for 2 hours, the mixture was concentrated under reduced pressure. The solution was then purified by Prep-HPLC to obtain (2S,4R)-1-((S)-2-(2-(4-((4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide trifluoroacetate (30 mg, yield 63%).

[0477] 1 H NMR (500MHz, DMSO-d6) δ9.00(s,1H),8.74(d,J=8.7Hz,1H),8.66(s,2H),8.42(d,J=7.8Hz,1H),7.44(d,J=8.4Hz,2H),7.38(d,J=8.3Hz,2H),6.8 8(d,J=1.8Hz,1H),6.81(d,J=6.8Hz,2H),6.56(d,J=1.9Hz,1H),5.11(s, 2H),4.92(p,J=7.1Hz,1H),4.57(d,J=9.1Hz,1H),4.45–4.41(m,2H),4.3 1(s,1H),4.26–4.22(m,4H),4.16–4.11(m,4H),4.01(t,J=16.5Hz,2H),3 .58–3.42(m,8H),3.34–3.26(m,1H),3.25–3.10(m,6H),3.09–2.95(m,2H ),2.46(s,3H),2.16(d,J=2.0Hz,6H),2.07–2.01(m,1H),1.96(s,2H),1. 86–1.77(m,1H),1.55(s,2H),1.38(d,J=7.0Hz,3H),0.98(s,9H).MS-ESI m / z=1050.5[M+1]+ .

[0478] Example 50

[0479] (2S,4R)-1-((S)-2-(4-(4-((4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)butamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0480] Referring to Example 49, ZZ24038 can be obtained using the same method.

[0481]

[0482] 1 H NMR (500MHz, DMSO-d6) δ8.99(s,1H),8.65(s,2H),8.39(d,J=7.8Hz,1H),8.07(d,J=9.3Hz,1H),7.44(d,J=8.3Hz,2H),7.38(d,J=8.3Hz,2H),6.8 7(d,J=1.8Hz,1H),6.81(d,J=6.8Hz,2H),6.56(d,J=1.9Hz,1H),5.11(s, 2H),4.92(p,J=6.9Hz,1H),4.53(d,J=9.3Hz,1H),4.42(t,J=8.1Hz,4H), 4.30(s,1H),4.24(s,4H),4.14(t,J=6.0Hz,4H),3.25–3.11(m,8H),3.04 (s,2H),2.94–2.84(m,2H),2.45(s,3H),2.30–2.24(m,1H),2.16(d,J=2. 0Hz,6H),2.06–1.94(m,4H),1.92–1.84(m,2H),1.80(ddd,J=13.0,8.8,4 .7Hz,1H),1.51–1.41(m,2H),1.38(d,J=7.0Hz,3H),0.95(s,9H).MS-ESI m / z=1078.5[M+1] + .

[0483] Example 51

[0484] (2S,4R)-1-((S)-2-(3-(4-((4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)propamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0485] Referring to Example 49, ZZ24041 can be obtained using the same method.

[0486]

[0487] 1 H NMR(500MHz,DMSO-d6)δ8.99(s,1H),8.68(s,2H),8.40(d,J=7.8Hz,1H),8.27( d,J=9.2Hz,1H),7.44(d,J=8.2Hz,2H),7.38(d,J=8.3Hz,2H),6.87(d,J=1.8Hz, 1H),6.81(d,J=6.8Hz,2H),6.56(d,J=1.9Hz,1H),5.11(s,2H),4.92(p,J=7.3Hz ,1H),4.54(d,J=9.3Hz,1H),4.41(t,J=8.2Hz,4H),4.30(s,1H),4.25(s,4H),4. 14(t,J=6.1Hz,4H),3.56(d,J=11.5Hz,4H),3.34–3.28(m,2H),3.25–3.15(m,4 H),3.13(s,2H),2.92(s,2H),2.74(dp,J=30.9,7.3Hz,4H),2.45(s,3H),2.16(d ,J=2.0Hz,6H),2.06–1.95(m,4H),1.80(ddd,J=13.1,8.8,4.7Hz,1H),1.49(d,J =6.8Hz,1H),1.45(d,J=13.4Hz,1H),1.38(d,J=7.0Hz,3H),0.96(s,9H).MS-ESI m / z = 1064.5 [M+1] + .

[0488] Example 52

[0489] (2S,4R)-1-((S)-2-(2-(2-(2-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0490]

[0491]

[0492] Step 1: Methyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate

[0493]

[0494] Diethylene glycol (2.5 g, 16.34 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). Sodium hydride (620 mg, 15.52 mmol) was added under ice bath conditions, and the mixture was stirred for 10 minutes. Methyl bromoacetate (2.6 g, 24.51 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was quenched by adding saturated ammonium chloride aqueous solution under ice bath conditions. Water was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the organic phase was separated and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography to obtain methyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate (862 mg, yield 30%).

[0495] Step 2: 2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)methyl acetate

[0496]

[0497] Methyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate (725 mg, 4.07 mmol) was dissolved in dichloromethane (40 mL), and p-toluenesulfonyl chloride (1.16 g, 6.11 mmol), triethylamine (1.7 mL, 12.21 mmol), and 4-dimethylaminopyridine (24 mg, 0.20 mmol) were added sequentially. The mixture was reacted overnight at room temperature, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain methyl 2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)acetate (822 mg, yield 61%).

[0498] Step 3: tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-(2-methoxy-2-oxoethoxy)ethoxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0499]

[0500] Methyl 2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)acetate (47 mg, 0.14 mmol), tert-butyl-6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (80 mg, 0.14 mmol), potassium carbonate (58 mg, 0.42 mmol), and potassium iodide (5 mg, 0.03 mmol) were dissolved in N,N-dimethylformamide. In (10 mL), the mixture was stirred at 80 °C for 12 hours, water was added, and the mixture was extracted three times with ethyl acetate. The mixture was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-(2-methoxy-2-oxyethoxy)ethoxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (68 mg, yield 66%).

[0501] Step 4: 2-(2-(2-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetic acid

[0502]

[0503] Tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-(2-methoxy-2-oxoethoxy)ethoxy)ethyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (55 mg, 0.08 mmol): dissolved in a mixed solution of tetrahydrofuran (2 mL), methanol (2 mL), and water (2 mL), with the addition of lithium hydroxide (9 mg, 0.23 mmol). The mixture was stirred at 40°C for 12 hours. The pH was adjusted to 3-4 with saturated citric acid aqueous solution. Ethyl acetate was added for extraction. The organic phase was separated and dried with anhydrous sodium sulfate. After concentration under reduced pressure, crude 2-(2-(2-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetic acid was used directly for the next reaction.

[0504] Step 5: tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-(2-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)ethoxy)ethylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0505]

[0506] 2-(2-(2-(4-(2-(6-(6-(tert-butoxycarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)-6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzis[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetic acid (54 mg, 0.08 mmol), HATU (40 mg, 0.11 mmol) and 4-dimethylaminopyridine (29 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (8 mL) and stirred at room temperature for 20 minutes. Then (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (33 mg) was added. 0.08 mmol), stirred for 2 hours at room temperature, water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by Prep-HPLC to obtain tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-(2-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)ethoxy)ethylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (46 mg, yield 59%).

[0507] Step 6: (2S,4R)-1-((S)-2-(2-(2-(2-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0508]

[0509] 32 mg (0.03 mmol) of tert-butyl 6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(4-(2-(2-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)ethoxy)ethylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was dissolved in dichloromethane (5 mL), and trifluoroacetic acid was added. 1 mL (13.4 mmol), stirred at room temperature for 2 hours, concentrated under reduced pressure, purified by Prep-HPLC, and freeze-dried to obtain (2S,4R)-1-((S)-2-(2-(2-(2-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (21 mg, yield 72%).

[0510] 1 H NMR (500MHz, DMSO-d6): δ8.99(s,1H),8.43(d,J=7.6Hz,1H),7.47(d,J=9.5Hz,1H),7.43(dd,J=8.2,4.6Hz,2H),7.33(d,J=8.2Hz,2H) ,6.89(s,1H),6.81(d,J=6.7Hz,2H),6.56(s,1H),5.11(s,2H),4.87(dt,J=14.4,7.1Hz,1H),4.57(d,J=9.6Hz,1H),4.48–4.33(m,3H), 4.31–4.22(m,5H),4.13(t,J=5.9Hz,4H),4.02(d,J=4.9Hz,2H),3.89–3.79(m,4H),3.75–3.54(m,8H),3.49–3.41(m,2H),3.36–3.10( m,4H),2.45(s,3H),2.14(d,J=1.1Hz,6H),2.07(dd,J=13.3,7.2Hz,1H),1.81–1.71(m,1H),1.33(d,J=7.0Hz,3H),0.94(s,9H).MS-ESI m / z=1041.5[M+1] + .

[0511] Example 53

[0512] (2S,4R)-1-((S)-2-(3-(2-(2-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)propamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0513] Referring to Example 52, ZC79039 can be obtained using the same method.

[0514]

[0515] 1 H NMR(500MHz,DMSO-d6):8.99(s,1H),8.37(d,J=7.8Hz,1H),7.89(d,J=9.4Hz,1 H),7.47–7.30(m,6H),7.03(s,1H),6.98(d,J=1.4Hz,1H),6.58(d,J=1.5Hz,1H) ,5.21(s,2H),4.90(dd,J=14.5,7.1Hz,1H),4.54(d,J=9.4Hz,1H),4.46–4.36( m,3H),4.26(d,J=18.1Hz,5H),4.13(t,J=6.0Hz,4H),4.01–3.04(m,18H,partly covered by the peakof water),2.66–2.34(m,7H),2.06–1.98(m,1H),1.83–1.75(m,1H),1.37(d,J=6.9Hz,3H),0.93(s,9H).MS-ESI m / z=1061.4[M+1] + .

[0516] Example 54

[0517] (2S,4R)-1-((S)-14-(tert-butyl)-1-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)-12-oxo-3,6,9-trioxa-13-azapentane-15-hydroxy)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0518] Referring to Example 52, ZC79043 can be obtained using the same method.

[0519]

[0520] 1 H NMR (500MHz, DMSO-d6): 8.99(s,1H),8.39(d,J=7.8Hz,1H),7.88(d,J=9.4Hz,1H),7.47–7.30(m,6H),7.05–6.96(m,2H),6.57(d,J= 1.7Hz,1H),5.21(s,2H),4.96–4.86(m,1H),4.53(d,J=9.4Hz,1H),4.45–4.36(m,3H),4.30–4.20(m,5H),4.19–3.70(m,8H,covered by the peak of water),3.66–3.45(m,12H),3.44–3.36(m,2H),3.26(d,J=11.1Hz,2H),3.15(t,J=12.1Hz,2H),2.60–2.29(m,7H,partly covered by the peak ofsolvent),2.06–1.97(m,1H),1.83–1.74(m,1H),1.37(d,J=7.0Hz,3H),0.92(s,9H).MS-ESI m / z=1105.5[M+1] + .

[0521] Example 55

[0522] (2S,4R)-1-((S)-2-(3-(2-(2-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)ethoxy)ethoxy)propamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0523] Referring to Example 52, ZC79122 can be obtained using the same method.

[0524]

[0525] 1H NMR (500MHz, DMSO-d6): δ9.00(s,1H),8.40(d,J=7.8Hz,1H),7.92(d,J=9.4Hz,1H ),7.44(d,J=8.2Hz,2H),7.37(d,J=8.3Hz,2H),6.88(d,J=1.4Hz,1H),6.81(d,J= 6.7Hz,2H),6.57(d,J=1.3Hz,1H),5.11(s,2H),4.95–4.87(m,1H),4.54(d,J=9.4 Hz,1H),4.45–4.36(t,J=8.1Hz,3H),4.30–4.22(m,5H),4.21–3.70(m,6H,partly covered by the peak of water),3.69–3.52(m,10H),3.45–3.35(m,2H),3.33–3.21(m,2H),3.16(t,J=12.0Hz,2H),2.61–2.34(m,7H,partly covered by the peak of solvent),2.16(d,J=1.2Hz,6H),2.08–1.99(m,1H),1.83–1.74(m,1H),1.37(d,J=7.0Hz,3H),0.93(s,9H).MS-ESI m / z=1055.5[M+1] + .

[0526] Example 56

[0527] (2S,4R)-1-((S)-14-(tert-butyl)-1-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)-12-oxo-3,6,9-trioxa-13-azapentane-15-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0528] Referring to Example 52, ZC79128 can be obtained using the same method.

[0529]

[0530] 1H NMR (500MHz, DMSO-d6): δ9.00(s,1H),8.40(d,J=7.8Hz,1H),7.90(d,J=9.4Hz,1H),7.44(d,J=8.2Hz,2H), 7.38(d,J=8.3Hz,2H),6.88(s,1H),6.81(d,J=6.7Hz,2H),6.57(s,1H),5.11(s,2H),4.97–4.86(m,1H),4.5 3(d,J=9.4Hz,1H),4.45–4.35(m,3H),4.31–4.21(m,5H),4.14(t,J=6.0Hz,4H),3.85–3.77(m,2H),3.70–3. 46(m,14H),3.45–3.36(m,2H),3.34–3.21(m,2H),3.15(t,J=12.2Hz,2H),2.61–2.30(m,7H,partlycovered by the peak of solvent),2.16(d,J=1.1Hz,6H),2.07–1.98(m,1H),1.83–1.73(m,1H),1.37(d,J=7.0Hz,3H),0.93(s,9H).MS-ESI m / z=1099.5[M+1] + .

[0531] Example 57

[0532] 4-((6-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0533]

[0534] Step 1: 2-(2,6-dioxopiperidin-3-yl)-4-((6-hydroxyhexyl)amino)isoindoline-1,3-dione

[0535]

[0536] 2-(2,6-dioxadipin-3-yl)-4-fluoroisoindoline-1,3-dione (1.18 g, 4.27 mmol), 6-amino-1-hexanol (500 mg, 4.27 mmol), and N,N-diisopropylethylamine (2.12 ml, 12.81 mmol) were dissolved in N,N-dimethylformamide and stirred at 90 °C for 12 hours. After cooling to room temperature, water was added, and the mixture was extracted three times with ethyl acetate. The extract was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain 2-(2,6-dioxadipin-3-yl)-4-((6-hydroxyhexyl)amino)isoindoline-1,3-dione (632 mg, yield 40%).

[0537] Step 2: 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)hexanal

[0538]

[0539] 2-(2,6-dioxopiperidin-3-yl)-4-((6-hydroxyhexyl)amino)isoindoline-1,3-dione (120 mg, 0.32 mmol) was dissolved in dichloromethane (10 mL), and Dys-Martin oxidant (204 mg, 0.48 mmol) was added. The mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexanal (58 mg, yield 49%).

[0540] Step 3: Tert-butyl 6-(6-chloro-4-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)piperazin-1-yl)-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0541]

[0542] 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)hexanal (34 mg, 0.09 mmol), tert-butyl-6-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (53 mg, 0.09 mmol) and glacial acetic acid (17 mg, 0.28 mmol) were dissolved in N,N-dimethylformamide (10 mL), stirred at room temperature for 30 minutes, and then sodium cyanoborohydride (35 mg, 0.09 mmol) was added. 55 mmol), stirred at room temperature for 3 hours, water was added to the reaction solution, extracted with ethyl acetate, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by Prep-HPLC to obtain tert-butyl 6-(6-chloro-4-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)piperazin-1-yl)-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (36 mg, yield 42%).

[0543] Step 4: 4-((6-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0544]

[0545] 24 mg (0.03 mmol) of tert-butyl 6-(6-chloro-4-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)piperazin-1-yl)-1-(4-fluoro-3,5-dimethylbenzyl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL, 13 mmol) was added. 4 mmol), stirred at room temperature for 2 hours, concentrated under reduced pressure and purified by Prep-HPLC, and freeze-dried to obtain 4-((6-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (15 mg, yield 71%).

[0546] 1H NMR (500MHz, DMSO-d6): δ11.12(s,1H),7.64–7.56(m,1H),7.11(d,J=8.6Hz,1H),7.04(d,J=7.0Hz,1H),6.87(s,1H), 6.81(d,J=6.7Hz,2H),6.60–6.51(m,2H),5.11(s,2H),5.06(dd,J=12.8,5.4Hz,1H),4.41(d,J=12.3Hz,2H),4.24(s, 4H),4.13(t,J=6.0Hz,4H),3.61(d,J=11.1Hz,2H),3.32(dd,J=12.3,6.3Hz,2H),3.25–3.02(m,6H),2.94–2.83(m,1H ),2.66–2.42(m,2H),2.16(d,J=0.8Hz,6H),2.02(dd,J=8.9,3.7Hz,1H),1.78–1.56(m,4H),1.46–1.30(m,4H).MS-ESI m / z=824.4[M+1] + .

[0547] Example 58

[0548] 4-((5-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0549] Referring to Example 57, ZC79147 can be obtained using the same method.

[0550]

[0551] 1H NMR (500MHz, DMSO) δ11.12(s,1H),7.60(dd,J=8.3,7.4Hz,1H),7.13(d,J=8.6Hz,1H),7.05(d ,J=7.0Hz,1H),6.88(s,1H),6.81(d,J=6.7Hz,2H),6.63–6.54(m,2H),5.11(s,2H),5.06(dd, J=12.8,5.4Hz),4.41(d,J=12.0Hz,2H),4.25(s,4H),4.13(t,J=5.9Hz,4H),3.61(d,J=11.1H z,2H),3.35(q,J=5.8Hz,2H),3.26–3.02(m,6H),2.94–2.83(m,1H),2.69–2.45(m,2H,partly covered by the peak of solvent),2.16(s,6H),2.03(dd,J=9.1,3.7Hz,1H),1.80–1.69(m,2H),1.68–1.59(m,2H),1.45–1.35(m,2H).MS-ESIm / z=810.4[M+1] + .

[0552] Example 59

[0553] 4-((8-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)octyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0554] Referring to Example 57, ZC57008 can be obtained using the same method.

[0555]

[0556] 1H NMR (500MHz, DMSO-d6): δ11.11(s,1H),7.63–7.55(m,1H),7.10(d,J=8.6Hz,1H),7.03(d,J=7 .0Hz,1H),6.88(s,1H),6.81(d,J=6.7Hz,2H),6.55(d,J=12.1Hz,2H),5.11(s,2H),5.05(dd,J =12.8,5.4Hz,1H),4.40(d,J=10.8Hz,2H),4.25(s,4H),4.14(t,J=5.9Hz,4H),3.60(d,J=11.1 Hz,2H),3.31(d,J=4.7Hz,2H),3.24–3.02(m,6H),2.95–2.83(m,1H),2.66–2.42(m,2H,partly covered by the peak of solvent),2.16(s,6H),2.06–1.99(m,1H),1.75–1.63(m,2H),1.62–1.53(m,2H),1.42–1.24(m,8H).MS-ESI m / z=852.4[M+1] + .

[0557] Example 60

[0558] 4-((10-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)decyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0559] Referring to Example 57, ZC57009 can be obtained using the same method.

[0560]

[0561] 1H NMR (500MHz, DMSO-d6): δ11.11(s,1H),7.62–7.54(m,1H),7.09(d,J=8.6Hz,1H),7.03(d,J=7 .0Hz,1H),6.87(s,1H),6.81(d,J=6.7Hz,2H),6.54(d,J=14.1Hz,2H),5.11(s,2H),5.05(dd,J =12.8,5.5Hz,1H),4.40(d,J=13.5Hz,2H),4.25(s,4H),4.13(t,J=5.9Hz,4H),3.60(d,J=11.3 Hz,2H),3.30(d,J=5.2Hz,2H),3.24–3.00(m,6H),2.94–2.82(m,1H),3.66–3.42(m,2H,partly covered by the peak of solvent),2.16(s,6H),2.06–1.99(m,1H),1.73–1.63(m,2H),1.62–1.53(m,2H),1.30(s,12H).MS-ESI m / z=880.4[M+1] + .

[0562] Example 61

[0563] 4-((9-(4-(6-chloro-1-(4-fluoro-3,5-dimethylbenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)nonyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0564] Referring to Example 57, ZC57010 can be obtained using the same method.

[0565]

[0566] 1H NMR (500MHz, DMSO-d6): δ11.10(s,1H),7.58(t,J=7.8Hz,1H),7.09(d,J=8.6Hz,1H),7.03( d,J=7.1Hz,1H),6.87(s,1H),6.81(d,J=6.8Hz,2H),6.54(d,J=13.4Hz,2H),5.11(s,2H),5. 05(dd,J=12.5,5.3Hz,1H),4.40(s,2H),4.24(s,4H),4.13(t,J=6.0Hz,4H),3.60(d,J=10. 8Hz,2H),3.35–3.25(m,2H),3.24–3.02(m,6H),2.95–2.83(m,1H),2.65–2.40(m,2H,partly covered by the peak of solvent),2.16(s,6H),2.06–1.98(m,1H),1.73–1.63(m,2H),1.62–1.53(m,2H),1.31(s,10H).MS-ESI m / z=866.4[M+1] + .

[0567] Example 62

[0568]

[0569] Step 1: 10-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-10-oxodecanoic acid

[0570]

[0571] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (100 mg, 0.23 mmol), sebacic acid (46 mg, 0.23 mmol), EDCI (52 mg, 0.27 mmol), HOAT (37 mg, 0.27 mmol), and N-methylmorpholine (46 mg, 0.45 mmol) were added to dichloromethane (4 mL) and N,N-dimethyl... In a mixed solvent of methylformamide (1 mL), the mixture was stirred overnight at room temperature, water was added, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by Prep-HPLC, and freeze-dried to obtain 10-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-10-oxodecanoic acid (46 mg, yield 33%).

[0572] Step 2: tert-butyl 6-(6-chloro-1-(3-chloro-4-fluorobenzyl)-4-(4-(9-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-9-oxonanoyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester

[0573]

[0574] 10-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-10-oxodecanoic acid (40 mg, 0.06 mmol), HATU (29 mg, 0.08 mmol), and N,N-diisopropylethylamine (25 mg, 0.19 mmol) were added to N,N-dimethylformamide (6 mL), and stirred at room temperature for 30 minutes. Then, tert-butyl-6-(6-chloro-1-(3-chloro-4-fluorobenzyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid was added. Salt (37 mg, 0.06 mmol) was stirred at room temperature for 2 hours, water was added, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by Prep-HPLC to give tert-butyl 6-(6-chloro-1-(3-chloro-4-fluorobenzyl)-4-(4-(9-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-9-oxonanoyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester (40 mg, yield 53%).

[0575] Step 3: (2S,4R)-1-((S)-2-(9-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)-9-oxoaminoamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0576]

[0577] 30 mg (0.03 mmol) of tert-butyl 6-(6-chloro-1-(3-chloro-4-fluorobenzyl)-4-(4-(9-((((S)-1-((2S,4R)-4-hydroxy-2-((((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-9-oxonanoyl)piperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid ester was added to dichloromethane (5 mL), followed by the addition of trifluoroacetic acid (0.5 g / mL). (mL, 6.73mmol), stirred at room temperature for 2 hours, concentrated under reduced pressure, purified by Prep-HPLC, and freeze-dried to obtain (2S,4R)-1-((S)-2-(9-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)-9-oxoaminoamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (17 mg, yield 61%).

[0578] 1 H NMR (500MHz, DMSO-d6): δ8.98(s,1H),8.37(d,J=7.8Hz,1H),7.78(d,J=9.4Hz,1H),7.50–7.31(m,6H),7.06–6.99(m,1H),6.90 (s,1H),6.48(s,1H),5.20(s,2H),4.95–4.88(m,1H),4.52(d,J=9.3Hz,1H),4.42(t,J=8.0Hz,1H),4.30–4.24(d,J=12.0Hz,5H) ,4.13(t,J=6.1Hz,4H),3.95–3.05(m,10H),2.65–2.62(m,1H),2.45(s,3H),2.35(dd,J=12.9,4.7Hz,3H),2.30–2.06(m,2H),2. 05–1.96(m,1H),1.84–1.75(m,1H),1.57–1.42(m,4H),1.37(d,J=7.0Hz,3H),1.25(d,J=15.9Hz,8H),0.98–0.89(m,9H).MS-ESI m / z=1071.5[M+1] + .

[0579] Example 63

[0580] (2S,4R)-1-((S)-2-(10-(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]heptane-2-yl)-1H-benzo[d]imidazol-4-yl)piperazin-1-yl)-10-oxododecanoamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0581] Referring to Example 62, ZC03140 can be obtained using the same method.

[0582]

[0583] 1 H NMR (500MHz, DMSO-d6): δ8.98(s,1H),8.37(d,J=7.8Hz,1H),7.78(d,J=9.4Hz,1H),7.50–7.31(m,6H),7.06–6.99(m,1H),6.90 (s,1H),6.48(s,1H),5.20(s,2H),4.95–4.88(m,1H),4.52(d,J=9.3Hz,1H),4.42(t,J=8.0Hz,1H),4.30–4.24(d,J=12.0Hz,5H) ,4.13(t,J=6.1Hz,4H),3.95–3.05(m,10H),2.65–2.62(m,1H),2.45(s,3H),2.35(dd,J=12.9,4.7Hz,3H),2.30–2.06(m,2H),2. 05–1.96(m,1H),1.84–1.75(m,1H),1.57–1.42(m,4H),1.37(d,J=7.0Hz,3H),1.25(d,J=15.9Hz,8H),0.98–0.89(m,9H).MS-ESI m / z=1085.5[M+1] + .

[0584] Example 64

[0585] N 1 -(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)benzyl)-N 6 -((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolid-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)hexamethylenediamide

[0586] Referring to Example 62, ZZ49053 can be obtained using the same method.

[0587]

[0588] 1 H NMR (500MHz, DMSO-d6) δ8.99(s,1H),8.47(s,2H),8.39–8.33(m,2H),7.97(d,J=7.9Hz,1H),7.80(d,J=9.3Hz,1H),7.46(dd,J=7.1,2. 1Hz,1H),7.44–7.41(m,2H),7.40–7.32(m,7H),7.30(d,J=2.0Hz,1H),7.11(d,J=7.5Hz,1H),5.29(s,2H),4.91(p,J=7.3Hz,1H),4.52 (d,J=9.3Hz,1H),4.42(t,J=8.1Hz,1H),4.37–4.26(m,7H),4.16–4.10(m,2H),2.45(s,3H),2.29(dd,J=14.1,7.5Hz,1H),2.21–2.09( m,4H),2.05–1.96(m,2H),1.79(ddd,J=12.9,8.5,4.7Hz,1H),1.49(dd,J=25.8,7.2Hz,6H),1.37(d,J=7.0Hz,4H),0.93(s,9H).MS-ESI m / z=1055.4[M+1] + .

[0589] Example 65

[0590] N 1 -(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)benzyl)-N 9 -((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolid-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)nonadiamide

[0591] Referring to Example 62, ZZ49111 can be obtained using the same method.

[0592]

[0593] MS-ESI m / z = 1092.4 [M+1] + .

[0594] Example 66

[0595] N 1 -(4-(6-chloro-1-(3-chloro-4-fluorobenzyl)-2-(2,6-diazaspiro[3.3]hept-2-yl)-1H-benzo[d]imidazol-4-yl)benzyl)-N 10 -((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolid-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)decadiamide

[0596]

[0597] Referring to Example 62, ZZ49057 can be obtained using the same method. MS-ESI m / z = 1106.5 [M+1] + .

[0598] Example 67

[0599] 4-((8-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazoline)-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0600]

[0601] Referring to Example 30, ZZ24160 was obtained using the same method.

[0602] 1H NMR (500MHz, DMSO-d6) δ13.99(s,1H),11.10(s,1H),9.91–9.73(m,1H),8.31(d,J=3.3Hz,1H),7.57(dd,J=8.5,7.1Hz,1H),7.09(d,J=8.6Hz,1H ),7.04(s,1H),7.01(d,J=7.1Hz,1H),6.85(d,J=7.4Hz,2H),6.75(s,1H),6.53(t,J=5.3Hz,2H),5.77–5.61(m,1H),5.04(dd,J=12.8,5.4Hz,1H ),4.61(d,J=12.7Hz,1H),4.17(t,J=6.3Hz,2H),3.97(d,J=13.3Hz,1H),3.30(q,J=6.6Hz,2H),3.26–3.11(m,2H),2.87(ddd,J=17.1,13.8,5.4 Hz,1H),2.67–2.53(m,7H),2.05–1.95(m,4H),1.89–1.80(m,3H),1.80– 1.73(m,1H),1.70–1.53(m,7H),1.53–1.44(m,2H),1.37(s,6H).MS-ESI m / z=871.4[M+1] + .

[0603] Example 68

[0604] 4-((11-((6-(1-acetylpiperidin-4-yl)-4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-2-methylquinazolin)-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0605]

[0606] Referring to Example 30, ZZ23001 was obtained using the same method.

[0607] 1H NMR (500MHz, DMSO-d6) δ14.05(s,1H),11.10(s,1H),9.83(d,J=7.9Hz,1H),8.32(d,J=3.3Hz,1H),7.63–7.53(m,1H),7.08(d,J=8.7Hz,1H),7.06 (s,1H),7.01(d,J=7.1Hz,1H),6.86(s,1H),6.85(s,1H),6.75(s,1H),6. 52(t,J=5.8Hz,2H),5.71(p,J=7.2Hz,1H),5.04(dd,J=12.7,5.4Hz,1H), 4.66–4.57(m,1H),4.16(t,J=6.2Hz,2H),3.98(d,J=13.6Hz,1H),3.28( q,J=6.6Hz,2H),3.24–3.11(m,2H),2.88(ddd,J=17.7,13.8,5.4Hz,1H), 2.66–2.55(m,7H),2.05–1.95(m,4H),1.89–1.74(m,3H),1.62(d,J=7.0H z,3H),1.60–1.51(m,2H),1.51–1.43(m,2H),1.39–1.25(m,14H).MS-ESI m / z=913.5[M+1] +

[0608] Example 69

[0609] 4-((11-(4-(4-((((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-11-oxoundecyl)amino)-2-(2,6-dioxoperidin-3-yl)isodihydroindole-1,3-dione

[0610] Step 1: tert-butyl(R)-(11-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-11-oxoundecyl)carbamate

[0611] (R)-N-(1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)-7-methoxy-2-methyl-6-(piperidin-4-yl)quinazolin-4-amine (50 mg, 0.11 mmol), 11-((tert-butoxycarbonyl)amino)undecanoic acid (33 mg, 0.11 mmol), and HATU (45.5 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (4 mL), and N,N-diisopropylethylamine (90 μL) was added under ice bath conditions. The mixture was kept warm and stirred for 4 hours. The reaction was quenched with water, extracted with ethyl acetate, and the organic phase was separated and washed three times with water. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative TLC (dichloromethane / methanol = 20 / 1) to obtain tert-butyl(R)-(11-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazoline-6-yl)piperidin-1-yl)-11-oxoundecyl)carbamate (50 mg, yield: 62%).

[0612] Step 2: (R)-11-amino-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)undecane-1-one

[0613]

[0614] 50 mg (67.3 mmol) of tert-butyl(R)-(11-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazoline-6-yl)piperidin-1-yl)-11-oxoundecyl)carbamate was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (1 mL) was added at room temperature. The mixture was stirred at room temperature for 30 minutes and concentrated under reduced pressure to obtain crude (R)-11-amino-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazoline-6-yl)piperidin-1-yl)undecyl-1-one (70 mg), which was directly used in the next step of the reaction.

[0615] Step 3: 4-((11-(4-(4-((((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-11-oxoundecyl)amino)-2-(2,6-dioxoperidin-3-yl)isodihydroindole-1,3-dione

[0616]

[0617] (R)-11-amino-1-(4-(4-((1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)undecane-1-one crude product (70 mg, 0.07 mmol) and 2-(2,6-dioxadiidine-3-yl)-4-fluoroisodihydroindole-1,3-dione (32 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (4 mL), and N,N-diisopropylethylamine (129 μL, 0.78 mmol) was added. The solution was heated to 100 °C. The mixture was stirred for 12 hours, then extracted with water and ethyl acetate. The organic phase was separated, washed three times with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative HPLC to obtain 4-((11-(4-(4-((((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-11-oxoundecyl)amino)-2-(2,6-dioxoperidol-3-yl)isodihydroindole-1,3-dione (30 mg, yield: 43%).

[0618] 1 H NMR (400MHz, DMSO-d6) δ14.09(s,1H),11.11(s,1H),9.85(d,J=8.0Hz,1H),8.32(s,1H),7.57(dd,J=8.6,7.1Hz,1H),7.10–7.04(m,2H), 7.01(d,J=7.0Hz,1H),6.85(d,J=7.0Hz,2H),6.75(s,1H),6.52(t,J=5.0Hz,1H),5.73(q,J=8.6,7.2Hz,1H),5.05(dd,J=12.7,5.4Hz,1H) ,4.63(d,J=12.8Hz,1H),3.99(s,4H),3.31–3.19(m,2H),3.13(t,J=12.1Hz,1H),2.88(td,J=16.4,15.2,5.2Hz,1H),2.70–2.61(m,1H), 2.58(s,4H),2.41–2.23(m,1H),2.00(t,J=7.5Hz,1H),1.91–1.72(m,1H),1.56(dd,J=40.4,16.3Hz,7H),1.24(d,J=10.5Hz,19H).MS-ESI m / z=899.4[M+1] +

[0619] Example 70

[0620] 4-((5-(4-(4-(((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-5-5-oxopentyl)amino)-2-(2,6-dioxadipinidin-3-yl)isodihydroindole-1,3-dione

[0621]

[0622] Referring to Example 69, ZZ23020 was obtained using the same method.

[0623] 1 H NMR (400MHz, DMSO-d6) δ14.06(s,1H),11.11(s,1H),9.84(d,J=7.8Hz,1H),8.32(d,J=2.8Hz,1H),7.57(td,J=7.9,3.6Hz,1H),7.11(dd,J=8. 7,2.6Hz,1H),7.07(s,1H),7.01(d,J=7.0Hz,1H),6.88–6.82(m,2H),6.75(s,1H),6.58(s,1H),5.77–5.66(m,1H),5.04(dd,J=12.8,5.4Hz,1H ),4.63(d,J=12.8Hz,1H),4.03(d,J=14.1Hz,1H),3.99(s,3H),3.33(s,2H),3.24(s,1H),3.18–3.09(m,1H),2.94–2.80(m,1H),2.68–2.62(m ,1H),2.58(s,3H),2.43–2.35(m,1H),1.99(q,J=6.9,6.4Hz,2H),1.80(dd,J=25.0,12.4Hz,2H),1.71–1.41(m,6H),1.32–1.17(m,6H).MS-ESI m / z=815.3[M+1] + .

[0624] Example 71

[0625] 5-((5-(4-(4-((((R)-1-(3-amino-5-(trifluoromethyl)phenyl)ethyl)amino)-7-methoxy-2-methylquinazolin-6-yl)piperidin-1-yl)-5-oxopentyl)amino)-2-(2,6-dioxoperidin-3-yl)isodihydroindole-1,3-dione

[0626]

[0627] Referring to Example 69, ZZ23021-B was obtained using the same method.

[0628] 1 H NMR (400MHz, DMSO-d6) δ14.03(s,1H),11.08(s,1H),9.84(s,1H),8.32(d,J=2.6Hz,1H),7.56(d,J=8.4Hz,1H),7. 14(s,1H),7.06(s,1H),6.95(s,1H),6.85(s,2H),6.84(d,J=2.7Hz,2H),6.75(s,1H),5.83–5.57(m,1H),5.02(dd, J=12.7,5.4Hz,1H),4.64(d,J=11.7Hz,1H),4.04(d,J=14.2Hz,2H),3.99(s,3H),3.17(d,J=6.0Hz,4H),2.85(d,J =12.3Hz,1H),2.73–2.55(m,6H),2.04–1.92(m,2H),1.89–1.73(m,1H),1.61(d,J=6.6Hz,6H),1.23(s,4H).MS-ESI m / z=815.3[M+1] + .

[0629] Example 72: Western blot determination of SOS1 protein degradation rate

[0630] Discard the supernatant of the NCI-H358 (human non-small cell lung cancer cells) cell culture medium after drug intervention. Wash twice with pre-cooled PBS, add an appropriate amount of RIPA lysis buffer (with added protease inhibitors and phosphatase inhibitors) to lyse the cells, collect the cell lysate into EP tubes using a cell scraper, sonicate 3-5 times (150W, 5s on, 10s off), centrifuge at 4℃ (12000r / min, 15min), and collect the supernatant, which is the total cell protein. Quantitatively detect the protein concentration using the BCA method. Dilute the protein with 5× protein loading buffer and denature at 100℃ for 5min. Total cellular proteins were separated by electrophoresis in 8% SDS-PAGE (upper gel 70V, 30 min; lower gel 120V, 60 min), transferred to a membrane (250mA, 60 min), blocked with 5% milk for 1 h, incubated overnight at 4°C with primary antibody (SOS1 (CST, 12409S), β-tubulin (CST, 15115S)), washed three times with TBST, incubated at room temperature for 1.5 h with secondary antibody (Anti-Rabbit (Promega, W4018)), washed three times with TBST, and developed after incubation with ECL chemiluminescence buffer. The relative gray values ​​of each band were quantified using Quick Quant in GeneSys software (DMSO group as 100%) to obtain the degradation rate of SOS1 by different concentrations of compounds. The compound concentrations were logarithmic in Graphpad Prism, and the degradation rate was analyzed using a four-parameter fitting algorithm in nonlinear regression, yielding the DC value of the compound degradation rate as a function of the logarithm of concentration. 50 Curve and derive DC 50 value.

[0631] The grayscale values ​​of each band were analyzed using GeneSys software to calculate the concentration DC of the protein degradation-targeting chimeric PROTACs when SOS1 protein degradation reached 50%. 50 .

[0632]

[0633]

[0634] Note: A (10~100nM), B (100~1000nM), C (>1000nM)

[0635] Figure 1 The compound ZC79070 in Example 45 shows that it can induce the degradation of SOS1 protein in NCI-H358 cells in a dose-dependent manner, and a significant degradation effect is produced when the compound concentration reaches 156 nM.

[0636] Figure 2The results show that compound ZZ02151 in Example 1 can induce the degradation of SOS1 protein in NCI-H358 cells in a dose-dependent manner, and a significant degradation effect is produced when the compound concentration reaches 78 nM.

[0637] Figure 3 The half-maximal degradation concentration (DC50) and maximum degradation extent of compound ZC79070 in Example 45 are shown. Compound ZC79070 can induce the degradation of SOS1 protein in NCI-H358 cells, with a half-maximal degradation concentration of 73.07 nM and a maximum degradation extent of 99%.

[0638] Example 73: CellTiter-Glo method for determining the inhibitory activity of PROTACs on tumor cell 3D proliferation

[0639] NCI-H358 (human non-small cell lung cancer cells) tumor cells in the logarithmic growth phase were seeded at 100 μl (1000 cells) per well in a 96-well ultra-low adsorption plate (ThermoFisher, 174925). Different concentrations of compounds were added, and the cells were cultured at 37°C and 5% CO2 for 7 days. Then, 100 μl of CellTiter-Glo reagent (Promega, G9682) was added to each well, mixed well, and 100 μl was transferred to an OptiPltae-96 white plate. The plate was read using the Luminescence mode on a Spark multi-plate reader. The inhibition rate was calculated as (1 - (compound well value - blank value) / (DMSO well value - blank value)) * 100 to obtain the inhibition rate of different compound concentrations. The logarithm of the compound concentration was taken in Graphpad Prism, and the IC50 of the compound inhibition rate with respect to the logarithm of the concentration was obtained by nonlinear regression with a four-parameter fitting algorithm. 50 Curve and derive IC 50 value.

[0640] Example Compound numbering <![CDATA[NCI-H358 IC 50 (nM)]]> 1 ZZ02151 A 8 ZZ02080 C 13 ZZ02102 C 21 ZZ77119 B 33 ZZ24011 C 37 ZZ77153 B 44 ZC79069 C 45 ZC79070 C 46 ZZ77115 C 47 ZZ77116 C 48 ZZ77117 C

[0641] Note: A (10–100 nM), B (100–200 nM), C (200–400 nM)

[0642] Figure 4 The results of the antiproliferative activity study of compound ZC79070 against H358 cells in Example 45 show that compound ZC79070 has antiproliferative activity against H358 cells, and its IC50 value is [not specified]. 50 The value is 337.65 nM.

[0643] Figure 5The results of the antiproliferative activity study of compound ZZ02151 against H358 cells in Example 1 show that compound ZZ02151 has antiproliferative activity against H358 cells, and its IC50 value is [not specified]. 50 The value is 7.61 nM.

[0644] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A protein degradation-targeting chimera of general structural formula such as I, or a pharmaceutically acceptable salt thereof: SOS1 protein ligand-linking group L-E3 ligase ligand (I), in, The protein degradation-targeting chimera is:

2. A pharmaceutical composition comprising: The protein degradation-targeting chimera of claim 1 or a pharmaceutically acceptable salt thereof, and Pharmaceutically acceptable carrier.

3. Use of the protein degradation targeting chimera of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-small cell lung cancer.

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